Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis

ABSTRACT

The present invention concerns methods and compositions for inhibiting angiogenesis and/or tumor growth, survival and/or metastasis. In particular embodiments, the methods and compositions may concern ligands against placenta growth factor (PlGF), such as BP-1, BP-2, BP-3 or BP-4. Some methods may comprise administering one or more PlGF ligands, alone or in combination with one or more other agents, such as chemotherapeutic agents, other anti-angiogenic agents, immunotherapeutic agents or radioimmunotherapeutic agents to a subject. The PlGF ligands are effective to inhibit angiogenesis, tumor cell motility, tumor metastasis, tumor growth and/or tumor survival. In certain embodiments, PlGF ligands may be administered to subjects to ameliorate other angiogenesis related conditions, such as macular degeneration. In some embodiments, PlGF expression levels may be determined by any known method to select those patients most likely to respond to PlGF targeted therapies.

RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119(e) ofprovisional U.S. Patent Application Ser. No. 60/728,292, filed on Oct.19, 2005, the entire text of which is incorporated herein by reference.

FEDERALLY FUNDED RESEARCH

The studies disclosed herein were supported in part by grant DAMD17-03-1-0758 from the U.S. Department of Defense (A.P.T.). The U.S.government may have certain rights to practice the subject invention.

BACKGROUND

1. Field of the Invention

The present invention concerns methods and compositions for inhibitingangiogenesis, particularly pathologic angiogenesis and/or growth andmetastasis of tumors. In particular embodiments, the compositions andmethods concern inhibitors targeted to placenta growth factor-2 (PlGF)and/or its receptor, Flt-1, including but not limited to peptides,antibodies, antibody fragments, humanized antibodies, chimericantibodies, antibody analogs, aptamers, organic compounds and/or anyother molecule or compound known in the art that may be used to inhibitPlGF-mediated angiogenesis and/or metastasis. In more particularembodiments, the inhibitors may be peptides isolated by phage displayagainst PlGF.

2. Description of Related Art

The high expression of angiogenic growth factors is necessary for thegrowth and development of diverse cancers (Cao et al., 1998, J ClinInvest 101:1055-1063). Among such angiogenic growth factors, highexpression of vascular endothelial growth factor (VEGF) has received themost attention with regard to being associated with increased malignancyand metastasis (Abdulrauf et al., 1998, J Neurosurg 88:513-52; Ahmed etal., 2000, Placenta 21 Suppl A, S16-24). However, the role of VEGF andother members of the VEGF family of growth factors in tumor recurrenceor metastatic growth has not been clearly established. Many treatments,including radiation, cytokines, and cytotoxic agents, owe some of theirtherapeutic activity to anti-angiogenic mechanisms, one of which is thedown-regulation of VEGF (Taylor et al., 2002a, Clin Cancer Res8:1213-1222; Jiang et al., 1999, Mol Carcinog 26:213-225; Machein etal., 1999, Neuropathol Appl Neurobiol 25:104-112). Yet, markedinhibition of VEGF does not always result in durable responses or evencure. This suggests that factors other than VEGF may be capable ofrestoring a blood supply to surviving tumor cells.

A need exists in the field for methods and compositions directed towardsthe effective inhibition of tumor angiogenesis and/or growth andmetastasis, as well as angiogenesis associated with other diseases.

SUMMARY OF THE INVENTION

The present invention fulfills an unresolved need in the art byproviding methods and compositions for inhibiting, suppressing, blockingand/or eliminating angiogenesis and/or tumor metastasis. In certainembodiments, the compositions and/or methods may concern ligands forplacenta growth factor-2 (PlGF), and/or ligands binding to both Flt-1and PlGF. Such ligands may include, but are not limited to, peptides,antibodies, antibody fragments, humanized antibodies, chimericantibodies, antibody analogs, aptamers, organic compounds and/or anyother molecules or compounds known in the art that are ligands for PlGF.

In various embodiments, the PlGF ligands may be peptides. Methods foridentifying peptides that bind to particular targets are well known inthe art, including for example phage display techniques as discussedbelow. In phage display, libraries of peptides expressed on the surfaceof phage, such as filamentous bacteriophage, are created and may bescreened by selection against the target(s) of interest. After one ormore rounds of screening (panning) against the target, phage containingdisplay peptides that bind to the target may be isolated and the peptidesequences determined, for example by sequencing of the peptide-encodingDNA inserts in the phage nucleic acid.

Other embodiments concern methods and/or compositions for treatingsubjects, such as subjects with cancer and/or with a condition relatedto angiogenesis (e.g., macular degeneration, etc.). Subjects mayinclude, but are not limited to, humans, animals, cats, dogs, cows,sheep, goats, horses, alpacas and mammals. The methods and compositionsmay comprise one or more PlGF ligands to be administered to a subject.In preferred embodiments, peptides identified as ligands by phagedisplay against PlGF may be administered. Administration may be by anyroute known in the art, such as oral, nasal, buccal, inhalational,rectal, vaginal or topical. Alternatively, administration may be byorthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal,intraarterial, intrathecal or intravenous injection. In preferredembodiments, the PlGF ligand (inhibitor) is orally administered. In morepreferred embodiments, the PlGF ligand may comprise the sequence of(binding protein) BP-1, BP-2, BP-3 or BP-4, as disclosed in the Examplesbelow.

The administration of PlGF ligands may be effective to inhibit oreliminate tumor metastasis, angiogenesis in solid tumors, tumor cellsurvival and/or tumor cell mobility. In preferred embodiments,administration of one or more PlGF ligands may prevent tumors frommetastasizing or may result in regression or growth inhibition ofexisting tumors. The skilled artisan will realize that one or more PlGFligands may be administered alone or alternatively in conjunction withother known therapeutic treatments for cancer and/or angiogenesis, suchas chemotherapy, radiation therapy, immunotherapy, anti-VEGF agentsand/or other known anti-angiogenesis agents and the like. In someembodiments, the PlGF ligands may be administered with a bispecificantibody, with one binding site for the PlGF ligand and a second bindingsite for a tumor antigen or other target. In other embodiments, PlGFligands may be covalently attached to or provided as a fusion proteinwith an antibody, antibody fragment, monoclonal antibody, Fc fragment,Fc-binding protein or antibody binding protein.

In alternative embodiments, PlGF ligands may be of use to treatangiogenesis related conditions besides cancer. Examples of conditionsrelated to angiogenesis include, but are not limited to, rheumatoidarthritis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, sarcoidosis, asthma, edema, pulmonary hypertension, formationand development of tumors, psoriasis, diabetic retinopathy, maculardegeneration, corneal graft rejection, neovascular glaucoma, myocardialangiogenesis, plaque neovascularization, restenosis, neointima formationafter vascular trauma, telangiectasia, hemophiliac joints, angiofibroma,fibrosis associated with chronic inflammation, lung fibrosis, deepvenous thrombosis and wound granulation.

In other alternative embodiments, the PlGF ligands disclosed herein maybe used as targeting peptides, for example by conjugation with a complexor therapeutic agents. PlGF ligands, such as BP-1, BP-2, BP-3 and/orBP-4, may be covalently or non-covalently attached to various moietiesby methods well known in the art, such as the use of covalentcross-linking reagents. Many such agents, such as carbodiimides,bisimidates, N-hydroxysuccinimide ester of suberic acid,dimethyl-3,3′-dithio-bispropionimidate, azidoglyoxal,1,5-difluoro-2,4-(dinitrobenzene) and other cross-linkers of use forproteins and/or peptides are known and may be used. In variousembodiments, PlGF ligands may be attached to antibodies, antibodyfragments, chemotherapeutic agents, anti-angiogenic agents,pro-apoptosis agents, liposomes incorporating such agents or any otherknown therapeutic compound.

In still other embodiments, the PlGF ligands may be used as adjuncts fordiagnosis and/or imaging purposes. For example, PlGF ligands may betagged with any known contrast or detection agent or may be detectedusing any known methodologies, such as ELISA, etc. The PlGF ligands maybe used ex vivo, for example by immunohistochemistry of tissue sections,to detect tumors or other tissues that express PlGF. Alternatively, PlGFligands may be administered to a subject for in vivo detection oftissues expressing PlGF. Such compositions and methods may be of use,for example, to detect or diagnose the presence of a PlGF-expressingtumor and/or to identify those subjects with an angiogenesis or cellmotility-related condition that might benefit from therapies targetedagainst PlGF.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of particularembodiments of the invention. The embodiments may be better understoodby reference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1. BP1 interacts with the heparin-binding region of Flt-1. Reagentswere added to Flt-1-coated wells as indicated in the lower part of theFigure. BP1 used in this experiment contains a linker and the FLAGepitope at its C-terminus. Peptide binding was assessed by probing forthe FLAG epitope. A: 1 μM BP1 only; B: 2.5/25 U/ml heparin only; C: 2.5U/ml heparin followed by 1 μM BP1; D: 1 μM BP1 followed by 2.5 U/mlheparin. Values shown are mean of duplicate wells±SD from 2 separateexperiments. *P<0.0002 for heparin followed by 1 μM BP1 (C) vs. BP1 only(A) (ANOVA).

FIG. 2. PlGF stimulates in vitro invasion of MDA-MB-231 cells. The graphrepresents the cumulative results±SD (n=3-5 experiments). Abbreviationsused in the graph: ‘Pl’ is PlGF; ‘VE’ is VEGF. *P<0.05 (ANOVA).

FIG. 3. BP1 inhibits MDA-MB-231 xenograft growth and metastasis. FIG.3A. Tumor volume vs. time, s.c. model. Results shown are from one of twoexperiments±SD (n=3-4 mice/treatment). *P<0.05 vs. mock-treated or CPA(change in tumor volume (ANOVA)). FIG. 3B. BP1 inhibited the formationof lung metastases in MDA-MB-231-bearing mice (s.c. model). Mice wereinjected subcutaneously with MDA-MB-231 cells. When tumors averaged0.120 cc in volume, treatment with peptides was commenced. One weekafter the last treatment primary tumors and lungs were removed formicroscopic examination. The Figure shows micrographs of lungs, H&Estain, from mock-treated (left) or BP1-treated (right) mice. Originalmagnification, 100×. “T” is adjacent to tumor.

FIG. 4. Exogenous PlGF stimulates increased tumor cell viability. Breasttumor cell lines were treated with PlGF (2 nM) or PlGF and thedesignated peptides (1-2 μM) under conditions of low serum concentration(1%). Twenty-four hours later viability of the cells was assessed byMTT. # denote P<0.04 of PlGF-treated cells compared to untreatedcontrols. Asterisks (*) denote P<0.03 compared to PlGF-only (ANOVA).FIG. 4A—MCF-7 human breast cancer cells. FIG. 4B—MDA-MB-468 human breastcancer cells. FIG. 4C—MDA-MB-231 human breast cancer cells.

FIG. 5. Treatment of mice bearing MDA-MB-468 xenografts with peptidesBP1 or BP3. Mice were treated every 3-4 days with 200 ug BP as describedin Methods. Tumor volume was measured 2×/week. *P<0.05 at days 10, 14,17 and 24 (change in tumor volume (ANOVA)). FIG. 5A—tumors treated withbinding peptide 1. FIG. 5B—tumors treated with binding peptide 3.

FIG. 6 Expression vector for hFc. The Figure provides a schematicdiagram of an exemplary vector (pdHL2-sCD20-hFc) that serves as thetemplate for constructing a vector (pdHL2-hFC) for expressing hFc. Therecombinant hFc may be chemically conjugated to BP1 as described inExample 4 or fused to hFc as described in Example 5.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “ligand” refers to any molecule, compound or composition that iscapable of binding to a target. For example, a PlGF ligand is a ligandthat can bind to PlGF. Binding capacity may be determined directly byany method known in the art, such as radioassay using labeled ligands ortargets, affinity chromatography, immunoprecipitation, dot blot, slotblot, Western blot, ELISA, microarray binding, etc. Alternatively,binding may be determined by indirect methods, such as panning ofpeptide-bearing phage against a target using a phage display technique.

Abbreviations used are:

-   -   BSA, bovine serum albumin;    -   ELISA, enzyme-linked immunosorbent assay;    -   FGF, fibroblast growth factor;    -   FGFR, fibroblast growth factor receptor;    -   FITC, fluorescein iso-thiocyanate;    -   flk-1, VEGF receptor II;    -   Flt-1, fms-like tyrosine kinase-1, VEGF receptor I;    -   FLAG, epitope-tagging system for detection of target proteins or        peptides;    -   HEC, human microvessel endothelial cells;    -   HRP, horseradish peroxidase;    -   IHC, immunohistochemistry;    -   MTT, 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium        bromide-based viability assay;    -   PBS, phosphate-buffered saline;    -   PlGF, placental growth factor;    -   RT, room temperature;    -   RTKs, receptor tyrosine kinases;    -   UT, untreated controls;    -   VEGF, vascular endothelial growth factor.        Placenta Growth Factor (PlGF) In Tumor Angiogenesis and        Metastasis

Various embodiments of the present invention concern compositions andmethods directed towards placenta growth factor (PlGF), a member of theVEGF family of growth factors. In certain embodiments, inhibitors(ligands) targeted to PlGF or targeted to PlGF and Flt-1 are of use toinhibit tumor angiogenesis and/or metastasis.

PlGF has a 53% identity with the platelet-derived growth factor-likeregion of VEGF (Maglione et al., 1991, Proc Natl Acad Sci USA88:9267-9271). It is produced as two main isoforms, PlGF-1 and -2. Theseare secreted as dimers composed of 132 or 153 amino acid monomers linkedin a head-to-tail fashion by disulfide bonds (Maglione et al., 1991),for a total weight of 46-50 kD. Three isoforms result from mRNA splicevariants, but only PlGF-1 and -2 are well characterized (Ahmed et al.,2000; Cao et al., 1997, Biochem Biophys Acta 235, 493-498).

First cloned from placenta (Maglione et al., 1991), PlGF is normallyexpressed by trophoblasts, normal thyroid, and during wound healing(Viglietto et al., 1995, Oncogene 11:1569-1579; Failla et al., 2000, JInvest Dermatol 115:388-395). In trophoblasts, PlGF expression isabrogated by hypoxic stress (Gleadle et al., Am. J. Physiol.268:C1362-8, 1995) but hypoxia-driven expression has been found in atleast one primary tumor (Yonekura et al., J. Biol. Chem. 274:35172-8,1999), in tumor xenografts (Taylor et al., Clin. Cancer Res.61:2696-703, 2001), and in fibroblasts (Green et al., Cancer Res.61:2696-703, 2001). Many malignancies express the PlGF receptor, Flt-1(Luttun et al., Nat. Med. 8:831-40, 2002).

PlGF is found in low levels in normal serum, but undergoes a 9-10-foldincrease in the serum of women without pre-eclampsia in late pregnancy(Helske et al., 2001, Mol Hum Reprod 7:205-210; Reuvekamp et al., 1999,Br J Obstet Gynaecol 106:1019-1022; Vuorela-Vepsalainen et al., 1999,Hum Reprod 14:1346-1351). PlGF has been detected in glioblastomas andmeningiomas, even though it is not expressed in normal brain tissue(Gleadle et al., 1995, Am J Physiol 268:C1362-1368; Donnini et al.,1999, J Pathol 189:66-71). It has also been detected in a variety ofother primary malignancies (Nomura et al., 1998, J. Neurooncol.40:123-30; Taylor et al., 2003a, Proc Am Assoc Cancer Res 44:4-5 [abstrR22]; Matsumoto et al., 2003, Anticancer Res 23:3767-3773; Chen et al.,2004, Cancer Lett 213:73-82; Wei et al., 2005, Gut 54, 666-672; Lacal etal., 2000, J Invest Dermatol 115, 1000-1007). PlGF expression has alsobeen observed associated with diabetic retinopathy with neovascularproliferation (Khaliq et al., 1998, Lab Invest. 78:109-16). Deletionexperiments with PlGF showed an inhibition of tumor growth and reductionof retinal neovascularization in animal models (Carmeliet et al., 2001,Nat. Med. 7:575-83).

PlGF-1, which lacks a heparin-binding domain, binds only to Flt-1 (alsotermed VEGFR1), while PlGF-2, which has a 21-amino-acid heparin-bindingdomain at the COOH terminus, binds both neuropilin-1 and Flt-1 (Migdalaet al., 1998, J Biol Chem 273:22272-22278; Hauser and Weich, 1993,Growth Factors 9:259-268). When bound to Flt-1, PlGF-2 is capable ofinducing differentiation, proliferation, and migration of endothelialcells (Landgren et al., 1998, Oncogene 16:359-367). In trophoblasts,expression of PlGF is abrogated by hypoxic stress (Gleadle et al.,1995), but hypoxia-driven expression has been found in at least oneprimary tumor (Yonekura et al., 1999, J Biol Chem 274:35172-35178), intumor xenografts (Taylor et al., 2004, Proc Am Assoc Cancer Res 45:981[abstr 4255]), and in fibroblasts (Green et al., 2001, Cancer Res61:2696-2703). Both PlGF-1 and -2 naturally form heterodimers with VEGF.The relative activity of the heterodimers may depend on the form of PlGFthat is bound to VEGF. Heterodimers consisting of the PlGF-1 isomer andVEGF have little or no activity in vitro or in vivo (Eriksson et al.,2002, Cancer Cell 1:99-108). On the other hand, PlGF-2/VEGF heterodimersare almost as potent as VEGF homodimers (Clauss et al., 1996, J BiolChem 271:17629-17634; Cao et al., 1996, J Biol Chem 271:3154-3162;DiSalvo et al., 1995, J Biol Chem 270:7717-7723).

The receptor for PlGF (and for VEGF), Flt-1, is a member of the tyrosinekinase family of receptors (RTKs). These are characterized byautophosphorylation of tyrosine residues in their cytoplasmic domainswhen bound by an activating ligand. Flt-1 has seven extracellularIg-like domains, and a split intracellular tyrosine kinase domain. Theinteraction of Flt-1 with PlGF lies mainly in the second extracellulardomain. The fourth domain of Flt-1 is associated with dimerization, andcontains a heparin-binding region that potentially brings Flt-1 and itsligand into closer association (Park and Lee, 1999, Biochem Biophys ResCommun 264:730-734). The anti-parallel PlGF dimer assures that thebinding domains are at opposite ends of the dimer, and thus, whenbinding, Flt-1 most likely brings two receptors into close association,linking them for activation (Fuh et al., 1998, J Biol Chem273:11197-11204; Wiesmann et al., 1997, Cell 91:695-704).

Heparin interactions with the heparin-binding domains of PlGF and Flt-1may be essential for full activation of Flt-1 by PlGF. The role ofheparin binding is well characterized in the fibroblast growth factor(FGF)-fibroblast growth factor receptor (FGFR) ligand-tyrosine kinasereceptor pair (Schlessinger et al., 2000, Molecular Cell 6:743-750). Inthis example, heparin makes multiple contacts with both the ligand andthe receptor, which increases FGF-FGFR binding to each other anddimerization of the receptors, a necessary step in activation. Thepresence of heparin-binding domains on the more active form of PlGF,PlGF-2, suggests that heparin may have a role in the activation of Flt-1by PlGF.

In our investigation of tumor recurrence, we detected PlGF production bysurviving tumor cells following cytotoxic treatment with radiolabeledantibodies (Taylor et al., 2002a; Taylor et al., 2003; Taylor et al.,2002b, Proc Am Assoc Cancer Res 43: 10-1 [abstr 51]), even when PlGFexpression was undetectable before treatment. It is already establishedthat PlGF is a survival factor for endothelial cells (Adini et al.,2002, Cancer Res 62:2749-2752), but its treatment-induced expression bytumor cells was an unexpected finding (Taylor et al., 2002a; Taylor etal., 2003b, Int J Cancer 105:158-169). Further investigation correlatedPlGF expression with enhanced recovery from cytotoxic treatment (Tayloret al., 2003b). Treatment-induced as well as constitutive expression bysome tumor cell lines was also found.

Hypoxia, a condition ubiquitous in tumors due to metabolic activity ortreatment, results in increased PlGF expression by keratinocytes andtumor cells (Ahmed et al., 2000; Taylor et al., 2002a; Taylor et al.,2003b). Some malignant cells express low levels of Flt-1 on theirsurface, yet the direct effects of signaling through the ligand-receptorare not well characterized. These findings suggested that PlGF mightexert effects on tumor cells and in the tumor environment. The presentdisclosure examined the direct effects of PlGF and Flt-1 on human breastcancer cells and xenografts.

Relative Roles of PlGF and VEGF in Angiogenesis

Both PlGF and VEGF bind to the Flt-1 receptor and both have beenreported to stimulate angiogenesis in tumor and normal tissues. Theeffect of VEGF binding to the Flt-1 receptor on angiogenesis has beenexamined (El-Mousawi et al., 2003, J Biol. Chem. 278:46681-91; U.S.Patent Application publication No. 20040266694). El-Mousawi et al.(2003) used a random 16-mer phage display system panned againstrecombinant Flt-1 to isolate several apparent VEGF-binding peptides. Thestrongest interaction was observed with a peptide designated V5.2.Addition of V5.2 to HUVEC and HCEC endothelial cells stimulated withVEGF or PlGF was reported to inhibit endothelial cell proliferation andwas also reported to inhibit VEGF-mediated migration of HCECs andVEGF-induced capillary formation in Matrigel™ (El-Mousawi et al., 2003).It was suggested that these effects of V5.2 were mediated throughbinding to the Flt-1 receptor (Id.). However, the domain of Flt-1 thatbound to V5.2 was not characterized by these authors, and it was notdetermined whether the V5.2 effects were antagonistic with heparinactivation of Flt-1. In various embodiments, where administration of aPlGF ligand in combination with another anti-angiogenic agent, such asan anti-VEGF agent, is contemplated, peptide V5.2 and similar VEGFinhibitors may be of use.

The roles of VEGF-A, VEGF-B and PlGF in angiogenesis were examined byMalik et al., “Redundant roles of VEGF-B and PlGF during selectiveVEGF-A blockade in mice,” Blood, published online Sep. 27, 2005. Theseauthors reported that mice lacking either VEGF-B or PlGF display onlyminor developmental defects. Inhibition of VEGF-A activity, compared toinhibition of VEGF-A, VEGF-B and PlGF, resulted in similar effects ongrowth and survival of neonatal mice, including a reduction invascularization. It was concluded that PlGF and VEGF-B do not compensatefor blockade of VEGF-A, indicating that VEGF-B and PlGF activation ofthe VEGFR-1 receptor plays a relatively minor role in postnataldevelopment and adult vascular homeostasis compared to VEGF-A activationof VEGFR-2. These results conflicted with other reports that antibodiesagainst VEGFR-1 were almost as effective as anti-VEGFR-2 antibodies ininhibiting xenograft tumor growth and angiogenesis (Carmeliet et al.,2001, Nat. Med. 7:575-83; Stefanik et al.,2001, J. Neurooncol.55:91-100). It was suggested that VEGF-B and PlGF may be involved inregulation of inflammatory events during pathologic angiogenesis inadults (Malik et al., 2005).

The skilled artisan will realize that the PlGF ligands disclosed hereinmay be used in combination with one or more VEGF inhibitors. VEGFinhibitors of potential use include Neovastat® (Falardeau et al., 2001,Semin. Oncol. 28:620-25), IM862 (Tupule et al., 2000, J. Clin. Oncol.18:716-23), Angiozyme™ (RPI-4610) (Weng and Usman, 2001, Curr. Oncol.Rep. 3:141-46), Bevacizumab (Gordon et al., 2001, J. Clin Oncol.19:843-50), Semaxanib (SU-5416) (Fong et al., 1999, Cancer Res. 59:99)and TNP-470 (Figg et al., 1997, Pharmocotherapy 17:91-97.

Therapeutic Treatment of Subjects by Inhibiting Angiogenesis

In various embodiments, the methods and compositions relating to PlGFligands that can block or inhibit PlGF-mediated angiogenesis findapplication in treatment of those conditions characterized bypathological angiogenesis. Examples of these conditions include, but arenot limited to, rheumatoid arthritis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, sarcoidosis, asthma, edema,pulmonary hypertension, formation and development of tumors, psoriasis,diabetic retinopathy, macular degeneration, corneal graft rejection,neovascular glaucoma, myocardial angiogenesis, plaqueneovascularization, restenosis, neointima formation after vasculartrauma, telangiectasia, hemophiliac joints, angiofibroma, fibrosisassociated with chronic inflammation, lung fibrosis, deep venousthrombosis, wound granulation, and tumor angiogenesis, growth andsurvival. Not all subjects with an angiogenesis- or cellmotility-related disease state will express PlGF in diseased tissues,and the skilled artisan will realize that in certain embodiments, exvivo and/or in vivo PlGF expression assays may be utilized to identifythose subjects who would benefit most from PlGF-targeted therapeuticintervention.

The skilled artisan will realize that anti-angiogenic therapies mayutilize one or more of the PlGF ligands disclosed herein, and in certainalternative embodiments may comprise the administration of otheranti-angiogenic agents known in the art, such as endostatin™,angiostatin, laminin peptides, fibronectin peptides (including ED-Bfibronectin), plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, paclitaxel,accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470,platelet factor 4 or minocycline.

The skilled artisan will also realize that within the context oftreating subjects with tumors, the PlGF ligands disclosed herein may beused in combination with any known cancer therapy, as discussed in moredetail below. Known cancer therapies include, but are not limited to,administration of chemotherapeutic agents, radiation therapy, surgicalexcision, localized hyperthermia, anti-tumor antibodies and otheranti-angiogenic agents.

Peptide Administration

Various embodiments of the claimed methods and/or compositions mayconcern one or more therapeutic peptides to be administered to asubject. Administration may occur by any route known in the art,including but not limited to oral, nasal, buccal, inhalational, rectal,vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial, intrathecal or intravenous injection.

Unmodified peptides administered orally to a subject can be degraded inthe digestive tract and depending on sequence and structure may exhibitpoor absorption across the intestinal lining. However, methods forchemically modifying peptides to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are well known (see, for example, Blondelle et al.,1995, Biophys. J. 69:604-11; Ecker and Crooke, 1995, Biotechnology13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY, VOL. 1, ed. Wollf, John Wiley & Sons; Goodman and Shao, 1996,Pure & Appl. Chem. 68:1303-08). Such methods can be performed onpeptides that bind to a selected target, such as PlGF. Methods forpreparing libraries of peptide analogs, such as peptides containingD-amino acids; peptidomimetics consisting of organic molecules thatmimic the structure of a peptide; or peptoids such as vinylogouspeptoids, have also been described and may be used to construct PlGFbinding peptides suitable for oral administration to a subject.

In certain embodiments, preparation and administration of peptidemimetics that mimic the structure of known PlGF ligands, such as BP-1,BP-2, BP-3 or BP-4, may be used within the scope of the claimed methodsand compositions. In such compounds, the standard peptide bond linkagemay be replaced by one or more alternative linking groups, such asCH₂—NH, CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methodsfor preparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103, eachincorporated herein by reference.) Peptide mimetics may exhibit enhancedstability and/or absorption in vivo compared to their peptide analogs.

Alternatively, therapeutic peptides may be administered by oral deliveryusing N-terminal and/or C-terminal capping to prevent exopeptidaseactivity. For example, the C-terminus may be capped using amide peptidesand the N-terminus may be capped by acetylation of the peptide. Peptidesmay also be cyclized to block exopeptidases, for example by formation ofcyclic amides, disulfides, ethers, sulfides and the like.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Feb. 3,2005, incorporated herein by reference). Another alternative would use acyclic peptide comprising the sequence of BP-1, BP-2, BP-3 or BP-4flanked by cysteine residues and comprised of all D-amino acids. Such aD-amino acid cyclic analog may have the same folded conformation as theL-form, which may be assessed by computer modeling studies usingtechniques known in the art. In preferred embodiments, the modifiedpeptides will exhibit PlGF binding in the nanomolar or lower range.Modified peptides may be assayed for PlGF binding by standard assays,such as ELISA. The skilled artisan will be aware that peptidemodification should be followed by testing for target binding activityto direct the course of peptide modification. In certain embodiments,peptides and/or proteins may be orally administered by co-formulationwith proteinase- and/or peptidase-inhibitors.

Other methods for oral delivery of therapeutic peptides are disclosed inMehta (“Oral delivery and recombinant production of peptide hormones,”June 2004, BioPharm International). The peptides are administered in anenteric-coated solid dosage form with excipients that modulateintestinal proteolytic activity and enhance peptide transport across theintestinal wall. Relative bioavailability of intact peptides using thistechnique ranged from 1% to 10% of the administered dosage. Insulin, amuch larger protein than the PlGF binding peptides disclosed herein, hasbeen successfully administered in dogs using enteric-coatedmicrocapsules with sodium cholate and a protease inhibitor (Ziv et al.,1994, J. Bone Miner. Res. 18 (Suppl. 2):792-94. Oral administration ofpeptides has been performed using acylcarnitine as a permeation enhancerand an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, seeMehta, 2004). Excipients of use for orally administered peptides maygenerally include one or more inhibitors of intestinalproteases/peptidases along with detergents or other agents to improvesolubility or absorption of the peptide, which may be packaged within anenteric-coated capsule or tablet (Mehta, 2004). The enteric coating isresistant to acid, allowing the peptide to pass through the stomach intothe intestine for absorption. Organic acids may be included in thecapsule to acidify the intestine and inhibit intestinal proteaseactivity once the capsule dissolves in the intestine (Mehta, 2004).Another alternative for oral delivery of peptides would includeconjugation to polyethylene glycol (PEG)-based amphiphilic oligomers,increasing absorption and resistance to enzymatic degradation (Solteroand Ekwuribe, 2001, Pharm. Technol. 6:110).

In still other embodiments, peptides such as PlGF binding peptides maybe modified for oral or inhalational administration by conjugation tocertain proteins, such as the Fc region of IgG1 (see Examples 3-7).Methods for preparation and use of peptide-Fc conjugates are disclosed,for example, in Low et al. (2005, Hum. Reprod. 20:1805-13) and Dumont etal. (2005, J. Aerosol. Med. 18:294-303), each incorporated herein byreference. Low et al. (2005) disclose the conjugation of the alpha andbeta subunits of FSH to the Fc region of IgG1 in single chain orheterodimer form, using recombinant expression in CHO cells. The Fcconjugated peptides were absorbed through epithelial cells in the lungor intestine by the neonatal Fc receptor mediated transport system. TheFc conjugated peptides exhibited improved stability and absorption invivo compared to the native peptides. It was also observed that theheterodimer conjugate was more active than the single chain form. Largerproteins, such as erythropoietin, may also be effectively delivered byinhalation using Fc conjugation (Dumont et al., 2005).

In alternative embodiments, therapeutic peptides may be administered byan inhalational route (e.g., Sievers et al., 2001, Pure Appl. Chem.73:1299-1303). Supercritical carbon dioxide aerosolization has been usedto generate nano or micro-scale particles out of a variety ofpharmaceutical agents, including proteins and peptides (Id.)Microbubbles formed by mixing supercritical carbon dioxide with aqueousprotein or peptide solutions may be dried at lower temperatures (25 to65° C.) than alternative methods of pharmaceutical powder formation,retaining the structure and activity of the therapeutic peptide (Id.) Insome cases, stabilizing compounds such as trehalose, sucrose, othersugars, buffers or surfactants may be added to the solution to furtherpreserve functional activity. The particles generated are sufficientlysmall to be administered by inhalation, avoiding some of the issues withintestinal proteases/peptidases and absorption across thegastrointestinal lining.

Dosages of therapeutic peptides to be administered to a subject mayvary, depending upon the pharmacodynamic characteristics, the mode androute of administration, age, weight and health of the subject, thenature and extent of symptoms, concurrent therapy and frequency oftreatment. Dosages of peptides may range between about 1 and 3000 mg per50 kg body weight, preferably 10 to 1000 mg/50kg, more preferably 25 to800 mg/50 kg. A dose of between 8 and 800 mg/50 kg may be administereddivided into 1 to 6 doses per day or in a sustained release form. In onestudy, a single 500 μg dose of peptide administered to human subjectsresulted in peak plasma concentrations ranging from about 150 to 600pg/ml within 90 to 180 minutes (Mehta, 2004).

Phage Display

Certain embodiments of the claimed compositions and/or methods concernbinding peptides and/or peptide mimetics of various protein targets,such as PlGF. PlGF binding peptides (BP) may be identified by any methodknown in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which isincorporated herein by reference, disclose methods for preparing a phagelibrary. The phage display technique involves genetically manipulatingbacteriophage so that small peptides can be expressed on their surface(Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993,Meth. Enzymol. 21:228-257).

The past decade has seen considerable progress in the construction ofphage-displayed peptide libraries and in the development of screeningmethods in which the libraries are used to isolate peptide ligands. Forexample, the use of peptide libraries has made it possible tocharacterize interacting sites and receptor-ligand binding motifs withinmany proteins, such as antibodies involved in inflammatory reactions orintegrins that mediate cellular adherence. This method has also beenused to identify novel peptide ligands that may serve as leads to thedevelopment of peptidomimetic drugs or imaging agents (Arap et al.,1998a, Science 279:377-380). In addition to peptides, larger proteindomains such as single-chain antibodies may also be displayed on thesurface of phage particles (Arap et al., 1998a).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998a, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807, incorporated hereinby reference.

Proteins and Peptides

A variety of polypeptides or proteins may be used within the scope ofthe claimed methods and compositions. In certain embodiments, theproteins may comprise antibodies or fragments of antibodies containingan antigen-binding site. In other embodiments, a short peptide ligand ofa target, such as PlGF, may be used for a variety of purposes, such asdetecting the presence of PlGF receptors on a cell, tissue or organ,inhibiting or blocking binding of PlGF to its receptor(s), facilitatingor activating binding of PlGF to its receptors, or mimicking part of thePlGF molecule or its receptor(s).

As used herein, a protein, polypeptide or peptide generally refers, butis not limited to, a protein of greater than about 200 amino acids, upto a full length sequence translated from a gene; a polypeptide ofgreater than about 100 amino acids; and/or a peptide of from about 3 toabout 100 amino acids. For convenience, the terms “protein,”“polypeptide” and “peptide” are used interchangeably herein.Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown on Table 4 below. TABLE 4Modified and Unusual Amino Acids Abbr. Amino Acid Aad 2-Aminoadipic acidBaad 3-Aminoadipic acid Bala β-alanine, β-Amino-propionic acid Abu2-Aminobutyric acid 4Abu 4-Aminobutyric acid, piperidinic acid Acp6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib 2-Aminoisobutyric acidBaib 3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelic acid Dpr2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn N-EthylasparagineHyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp4-Hydroxyproline Ide Isodesmosine AIle allo-Isoleucine MeGlyN-Methylglycine, sarcosine MeIle N-Methylisoleucine MeLys6-N-Methyllysine MeVal N-Methylvaline Nva Norvaline Nle Norleucine OrnOrnithine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes, such as the PlGF gene,have been previously disclosed and may be found at computerizeddatabases known to those of ordinary skill in the art. One such databaseis the National Center for Biotechnology Information's Genbank andGenPept databases (www.ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

Peptide Mimetics

Another embodiment for the preparation of polypeptides is the use ofpeptide mimetics. Mimetics are peptide-containing molecules that mimicelements of protein secondary structure. See, for example, Johnson etal, “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto etal., Eds., Chapman and Hall, New York (1993), incorporated herein byreference. The rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains so as to facilitate molecular interactions, such as those ofantibody and antigen. A peptide mimetic is expected to permit molecularinteractions similar to the natural molecule. These principles may beused to engineer second generation molecules having many of the naturalproperties of the binding peptides disclosed herein, but with altered orimproved characteristics, such as increased absorption across thestomach or intestine and/or improved stability or activity in vivo.

Fusion Proteins

Various embodiments may concern fusion proteins. These moleculesgenerally have all or a substantial portion of a peptide, linked at theN— or C-terminus, to all or a portion of a second polypeptide orprotein. For example, fusions may employ leader sequences from otherspecies to permit the recombinant expression of a protein in aheterologous host. Another useful fusion includes the addition of animmunologically active domain, such as an antibody epitope. Yet anotheruseful form of fusion may include attachment of a moiety of use forpurification, such as the FLAG epitope (Prickett et al., 1989,Biotechniques 7:580-589; Castrucci et al., 1992, J Virol 66:4647-4653).Methods of generating fusion proteins are well known to those of skillin the art. Such proteins may be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding a first protein or peptide to a DNA sequence encodinga second peptide or protein, followed by expression of the intact fusionprotein.

Protein Purification

In some embodiments a protein or peptide may be isolated or purified.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.A particularly efficient method of purifying peptides is fast proteinliquid chromatography (FPLC) or even HPLC.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low-pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix, such as a magneticbead (Dynal) or a Sepharose or Sephadex bead (Pharmacia). The columnmaterial is then able to specifically adsorb the substance from thesolution. Elution occurs by changing the conditions to those in whichbinding will not occur (e.g., altered pH, ionic strength, temperature,etc.). The matrix should be a substance that itself does not adsorbmolecules to any significant extent and that has a broad range ofchemical, physical and thermal stability. The ligand should be coupledin such a way as to not affect its binding properties. The ligand shouldalso provide relatively tight binding. And it should be possible toelute the substance without destroying the sample or the ligand.

Synthetic Peptides

Proteins or peptides may be synthesized, in whole or in part, insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, (1984, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co.); Tam et al., (1983, J. Am. Chem. Soc., 105:6442);Merrifield, (1986, Science, 232: 341-347); and Barany and Merrifield(1979, The Peptides, Gross and Meienhofer, eds., Academic Press, NewYork, pp. 1-284). Short peptide sequences, usually from about 6 up toabout 35 to 50 amino acids, can be readily synthesized by such methods.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of interest is inserted intoan expression vector, transformed or transfected into an appropriatehost cell, and cultivated under conditions suitable for expression.

Antibodies

Various embodiments may concern antibody ligands for a target, such asanti-PlGF antibodies. The term “antibody” is used herein to refer to anyantibody-like molecule that has an antigen binding region, and includesantibody fragments such as Fab′, Fab, F(ab′)₂, single domain antibodies(DABs), Fv, scFv (single chain Fv), and the like. Techniques forpreparing and using various antibody-based constructs and fragments arewell known in the art. Means for preparing and characterizing antibodiesare also well known in the art (See, e.g., Harlowe and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory).Antibodies of use may also be commercially obtained from a wide varietyof known sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.).

Polyclonal Antibodies

Polyclonal antibodies may be prepared by immunizing an animal with animmunogen and collecting antisera from that immunized animal. A widerange of animal species may be used for the production of antisera.Typically an animal used for production of antisera is a non-humananimal, for example, rabbits, mice, rats, hamsters, pigs or horses.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, may be prepared usingconventional immunization techniques. A composition containing antigenicepitopes may be used to immunize one or more experimental animals, suchas a rabbit or mouse, which will then proceed to produce specificantibodies. Polyclonal antisera may be obtained, after allowing time forantibody generation, simply by bleeding the animal and preparing serumsamples from the whole blood.

A given composition may vary in its immunogenicity. It is oftennecessary, therefore, to boost the host immune system, as may beachieved by coupling an immunogen to a carrier. Exemplary and preferredcarriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin(BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbitserum albumin also may be used as carriers. Means for conjugating anantigen to a carrier protein are well known and include cross-linkerssuch as glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester,carbodiimide and bis-biazotized benzidine.

The immunogenicity of a particular immunogen composition may be enhancedby the use of non-specific stimulators of the immune response, known asadjuvants. Exemplary and preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes may be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection also may be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate monoclonal antibodies.

Monoclonal Antibodies

Monoclonal antibodies may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265.Typically, this technique involves immunizing a suitable animal with aselected immunogen composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Cells from rodents such as mice and rats are preferred. Mice aremore preferred, with the BALB/c mouse being most preferred as this ismost routinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of the animal with the highest antibody titerwill be removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B-lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art. For example, where the immunized animal is a mouse,one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one mayuse R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON—HMy2 and UC729-6 are all useful in connection with cellfusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus, and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, have been described. The use ofelectrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

A preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two wk. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three wk) for the desired reactivity. The assay should be sensitive,simple and rapid, such as radioimmunoassays, enzyme immunoassays,cytotoxicity assays, plaque assays, dot immunobinding assays, and thelike.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines also could be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation, and variouschromatographic methods such as HPLC or affinity chromatography.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments may be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment may be further cleaved using a thiol reducing agent and,optionally, a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5 S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.)

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, an anti-PlGF mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

In another embodiment, an antibody may be a human monoclonal antibody.Such antibodies are obtained from transgenic mice that have beenengineered to produce specific human antibodies in response to antigenicchallenge. In this technique, elements of the human heavy and lightchain locus are introduced into strains of mice derived from embryonicstem cell lines that contain targeted disruptions of the endogenousheavy chain and light chain loci. The transgenic mice can synthesizehuman antibodies specific for human antigens, and the mice can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described by Green et al.,Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), andTaylor et al., Int. Immun. 6:579 (1994).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique, as discussed above, maybe used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005,Genet. Mol. Res. 4:126-40, incorporated herein by reference). Humanantibodies may be generated from normal humans or from humans thatexhibit a particular disease state, such as cancer (Dantas-Barbosa etal., 2005). The advantage to constructing human antibodies from adiseased individual is that the circulating antibody repertoire may bebiased towards antibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and ε chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, as discussed above. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. A non-limiting example ofsuch a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol.Methods 231:11-23, incorporated herein by reference) from Abgenix(Fremont, Calif.). In the XenoMouse® and similar animals, the mouseantibody genes have been inactivated and replaced by functional humanantibody genes, while the remainder of the mouse immune system remainsintact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled, for example, to PlGFligands by chemical cross-linking or other known methodologies.Transgenically produced human antibodies have been shown to havetherapeutic potential, while retaining the pharmacokinetic properties ofnormal human antibodies (Green et al., 1999). The skilled artisan willrealize that the claimed compositions and methods are not limited to useof the XenoMouse® system but may utilize any transgenic animal that hasbeen genetically engineered to produce human antibodies.

Bi-Specific Antibodies and Conjugates

In certain embodiments, the PlGF ligands disclosed herein may be used incombination with another molecule attached to the ligand. Attachment maybe either covalent or non-covalent. In some embodiments, a PlGF ligandmay be attached to a bi-specific antibody, i.e., an antibody that hastwo different binding sites, one for the PlGF ligand and another for adisease-related target antigen. Any disease or condition relating toangiogenesis, cancer, metastasis or cell motility may be targeted,including but not limited to, primary cancer, metastatic cancer,hyperplasia, rheumatoid arthritis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, sarcoidosis, asthma, edema, pulmonaryhypertension, formation and development of tumor tissue, psoriasis,diabetic retinopathy, macular degeneration, corneal graft rejection,neovascular glaucoma, myocardial angiogenesis, plaqueneovascularization, restenosis, neointima formation after vasculartrauma, telangiectasia, hemophiliac joints, angiofibroma, fibrosisassociated with chronic inflammation, lung fibrosis, deep venousthrombosis and wound granulation. Methods for construction and use ofbi-specific and multi-specific antibodies are disclosed, for example, inU.S. Patent Application Publication No. 20050002945, filed Feb. 11,2004, the entire text of which is incorporated herein by reference.

Where the bi-specific antibody is targeted in part against atumor-associated antigen, it is anticipated that any type of tumor andany type of tumor antigen may be so targeted. Exemplary types of tumorsthat may be targeted include acute lymphoblastic leukemia, acutemyelogenous leukemia, biliary cancer, breast cancer, cervical cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectalcancer, endometrial cancer, esophageal, gastric, head and neck cancer,Hodgkin's lymphoma, lung cancer, medullary thyroid, non-Hodgkin'slymphoma, ovarian cancer, pancreatic cancer, glioma, melanoma, livercancer, prostate cancer, and urinary bladder cancer. Preferred aretumors that have constitutive expression of PlGF, or which can bestimulated to produce PlGF.

Tumor-associated antigens that may be targeted include, but are notlimited to, A3, antigen specific for A33 antibody, BrE3-antigen, CD1,CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45,CD74, CD79a, CD80, HLA-DR, NCA 95, NCA90, HCG and its subunits, CEA(CEACAM-5), CEACAM-6, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733,HER2/neu, hypoxia inducible factor (HIF), KC4-antigen, KS-1-antigen,KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3,MUC4, PAM-4-antigen, PSA, PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6,IL-8, insulin growth factor-1 (IGF-1), Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, VEGF, 17-1A-antigen, an angiogenesismarker (e.g., ED-B fibronectin), an oncogene marker, an oncogeneproduct, and other tumor-associated antigens. Recent reports on tumorassociated antigens include Mizukami et al., (2005, Nature Med.11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48);Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44) and Ren et al.(2005, Ann. Surg. 242:55-63), each incorporated herein by reference.

A variety of recombinant methods can be used to produce bi-specificantibodies and antibody fragments. For example, bi-specific antibodiesand antibody fragments can be produced in the milk of transgeniclivestock. (See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147,1998; U.S. Pat. No. 5,827,690, each incorporated herein by reference.)Two DNA constructs are prepared which contain, respectively, DNAsegments encoding paired immunoglobulin heavy and light chains. Thefragments are cloned into expression vectors which contain a promotersequence that is preferentially expressed in mammary epithelial cells.Examples include, but are not limited to, promoters from rabbit, cow andsheep casein genes, the cow alpha-lactoglobulin gene, the sheepbeta-lactoglobulin gene and the mouse whey acid protein gene.Preferably, the inserted fragment is flanked on its 3′ side by cognategenomic sequences from a mammary-specific gene. This provides apolyadenylation site and transcript-stabilizing sequences. Theexpression cassettes are coinjected into the pronuclei of fertilized,mammalian eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the progeny are screened forthe presence of both transgenes by Southern analysis. In order for theantibody to be present, both heavy and light chain genes must-beexpressed concurrently in the same cell. Milk from transgenic females isanalyzed for the presence and functionality of the antibody or antibodyfragment using standard immunological methods known in the art. Theantibody can be purified from the milk using standard methods known inthe art.

Pre-Targeting

One strategy for use of bi-specific antibodies includes pretargetingmethodologies, in which an effector molecule, such as an anti-angiogenicor anti-tumor PlGF ligand, is administered to a subject after abi-specific antibody has been administered. The bi-specific antibody,which would include a binding site for the PlGF ligand and one for thediseased tissue, localizes to the diseased tissue and increases thespecificity of localization of the effector PlGF ligand to the diseasedtissue (U.S. Patent Application No. 20050002945). Because the effectormolecule may be cleared from circulation much more rapidly than thebi-specific antibody, normal tissues may have a decreased exposure tothe effector molecule when a pretargeting strategy is used than when theeffector molecule is directly linked to the disease targeting antibody.

Pretargeting methods have been developed to increase thetarget:background ratios of detection or therapeutic agents. Examples ofpre-targeting and biotin/avidin approaches are described, for example,in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl.Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehret al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Ser. No. 09/597,580; U.S. Ser. No.10/361,026; U.S. Ser. No. 09/337,756; U.S. Ser. No. 09/823,746; U.S.Ser. No. 10/116,116; U.S. Ser. No. 09/382,186; U.S. Ser. No. 10/150,654;U.S. Pat. No. 6,090,381; U.S. Pat. No. 6,472,511; U.S. Ser. No.10/114,315; U.S. Provisional Application No. 60/386,411; U.S.Provisional Application No. 60/345,641; U.S. Provisional Application No.60/3328,835; U.S. Provisional Application No. 60/426,379; U.S. Ser. No.09/823,746; U.S. Ser. No. 09/337,756; and U.S. Provisional ApplicationNo. 60/342,103, all of which are incorporated herein by reference.

In certain embodiments, bispecific antibodies and targetable constructsmay be of use in treating and/or imaging normal or diseased tissue andorgans, for example using the methods described in U.S. Pat. Nos.6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093;5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119;5,101,827; and 4,735,210, each incorporated herein by reference.Additional methods are described in U.S. application Ser. No. 09/337,756filed Jun. 22, 1999 and in U.S. application Ser. No. 09/823,746, filedApr. 3, 2001.

Targeting Peptides

In other embodiments, peptide PlGF ligands such as BP-1, BP-2, BP-3 orBP-4 may be used as targeting peptides for delivery of one or moreagents to a diseased tissue. In such case, the agent may be covalentlyor non-covalently attached to the PlGF binding peptide. Agents ofpotential use include drugs, prodrugs, toxins, enzymes,oligonucleotides, radioisotopes, immunomodulators, cytokines, hormones,binding molecules, lipids, polymers, micelles, liposomes, nanoparticles,or combinations thereof. Exemplary therapeutic agents and methods of useare disclosed in U.S. Patent Publication Nos. 20050002945, 20040018557,20030148409 and 20050014207, each incorporated herein by reference.

In exemplary embodiments, agents of use may comprise one or more ofaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, anantisense oligonucleotide, an interference RNA, or a combinationthereof.

Aptamers

In certain embodiments, a PlGF ligand of use may be an aptamer. Methodsof constructing and determining the binding characteristics of aptamersare well known in the art. For example, such techniques are described inU.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, each incorporatedherein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the PlGF-binding sequences of aptamers may beflanked by primer-binding sequences, facilitating the amplification ofthe aptamers by PCR or other amplification techniques. In a furtherembodiment, the flanking sequence may comprise a specific sequence thatpreferentially recognizes or binds a moiety to enhance theimmobilization of the aptamer to a substrate.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20 C) and R′ isalkyl (1-20 C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

The aptamers used as starting materials in the process of the inventionto determine specific binding sequences may be single-stranded ordouble-stranded DNA or RNA. In a preferred embodiment, the sequences aresingle-stranded DNA, which is less susceptible to nuclease degradationthan RNA. In preferred embodiments, the starting aptamer will contain arandomized sequence portion, generally including from about 10 to 400nucleotides, more preferably 20 to 100 nucleotides. The randomizedsequence is flanked by primer sequences that permit the amplification ofaptamers found to bind to the target. For synthesis of the randomizedregions, mixtures of nucleotides at the positions where randomization isdesired may be added during synthesis.

Methods for preparation and screening of aptamers that bind toparticular targets of interest are well known, for example U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163, each incorporated by reference.The technique generally involves selection from a mixture of candidateaptamers and step-wise iterations of binding, separation of bound fromunbound aptamers and amplification. Because only a small number ofsequences (possibly only one molecule of aptamer) corresponding to thehighest affinity aptamers exist in the mixture, it is generallydesirable to set the partitioning criteria so that a significant amountof aptamers in the mixture (approximately 5-50%) are retained duringseparation. Each cycle results in an enrichment of aptamers with highaffinity for the target. Repetition for between three to six selectionand amplification cycles may be used to generate aptamers that bind withhigh affinity and specificity to the target, such as PlGF.

Methods of Disease Tissue Detection, Diagnosis and Imaging

Protein Based In Vitro Diagnosis

The present invention contemplates the use of PlGF ligands, includingPlGF binding peptides, PlGF fusion proteins, PlGF antibodies orfragments, bi-specific antibodies and antibody fragments, to screenbiological samples in vitro and/or in vivo for the presence of the PlGFantigen. In exemplary immunoassays, the PlGF antibody, fusion protein,or fragment thereof may be utilized in liquid phase or bound to asolid-phase carrier, as described below. In preferred embodiments,particularly those involving in vivo administration, the PlGF antibodyor fragment thereof is humanized. Also preferred, the PlGF antibody orfragment thereof is fully human. Still preferred, the PlGF fusionprotein comprises a humanized or fully human PlGF antibody. The skilledartisan will realize that a wide variety of techniques are known fordetermining levels of expression of a particular gene and any such knownmethod, such as immunoassay, RT-PCR, mRNA purification and/or cDNApreparation followed by hybridization to a gene expression assay chipmay be utilized to determine levels of PlGF expression in individualsubjects and/or tissues.

One example of a screening method for determining whether a biologicalsample contains the PlGF antigen is radioimmunoassay (RIA). For example,in one form of RIA, the substance under test is mixed with PlGF MAb inthe presence of radiolabeled PlGF antigen. In this method, theconcentration of the test substance will be inversely proportional tothe amount of labeled PlGF antigen bound to the MAb and directly relatedto the amount of free, labeled PlGF antigen. Other suitable screeningmethods will be readily apparent to those of skill in the art.

Alternatively, in vitro assays may be performed in which a PlGF ligand,anti-PlGF antibody, fusion protein, or fragment thereof is bound to asolid-phase carrier. For example, MAbs can be attached to a polymer,such as aminodextran, in order to link the MAb to an insoluble supportsuch as a polymer-coated bead, a plate or a tube.

The presence of the PlGF antigen in a biological sample may bedetermined using an enzyme-linked immunosorbent assay (ELISA). In thedirect competitive ELISA, a pure or semipure antigen preparation isbound to a solid support that is insoluble in the fluid or cellularextract being tested and a quantity of detectably labeled solubleantibody, antibody fragment or PlGF ligand is added to permit detectionand/or quantitation of the binary complex formed between solid-phaseantigen and labeled PlGF binding molecule.

A sandwich ELISA requires small amounts of antigen, and the assay doesnot require extensive purification of the antigen. Thus, the sandwichELISA is preferred to the direct competitive ELISA for the detection ofan antigen in a clinical sample. See, for example, Field et al.,Oncogene 4:1463 (1989); Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a sandwich ELISA, a quantity of unlabeled MAb or antibody fragment(the “capture antibody”) is bound to a solid support, the test sample isbrought into contact with the capture antibody, and a quantity ofdetectably labeled soluble antibody (or antibody fragment) is added topermit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. An antibodyfragment is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,and the like. In the present context, an antibody fragment is a portionof a PlGF MAb that binds to an epitope of the PlGF antigen. The term“antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker. Anantibody fusion protein is a recombinantly produced antigen-bindingmolecule in which two or more of the same or different single-chainantibody or antibody fragment segments with the same or differentspecificities are linked. The fusion protein may comprise a singleantibody component, a multivalent or multi specific combination ofdifferent antibody components or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment conjugated to a diagnostic/detection and/or atherapeutic agent. The term PlGF antibody includes humanized, human andmurine antibodies, antibody fragments thereof, immunoconjugates andfragments thereof and antibody fusion proteins and fragments thereof.

Methods of performing a sandwich ELISA are well-known. See, for example,Field et al., supra, Spandidos et al., supra, and Moore et al.,“Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAK System,”in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 273-281 (The HumanaPress, Inc. 1992). The skilled artisan will realize that an assaysimilar to a sandwich ELISA may be performed by substituting PlGF ligandfor either the first unlabeled antibody or the second labeled antibody.

In a sandwich ELISA, the soluble antibody or antibody fragment must bindto a PlGF epitope that is distinct from the epitope recognized by thecapture antibody. The sandwich ELISA can be performed to ascertainwhether the PlGF antigen is present in a biopsy sample. Alternatively,the assay can be performed to quantitate the amount of PlGF antigen thatis present in a clinical sample of body fluid. The quantitative assaycan be performed by including dilutions of purified PlGF antigen.

In other embodiments, Western blot analysis may be used to detect andquantify the presence of PlGF in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesor ligands that specifically bind PlGF. The anti-PlGF antibodies orligands specifically bind to PlGF on the solid support. These antibodiesor ligands may be directly labeled or alternatively may be subsequentlydetected using labeled secondary antibodies that specifically bind tothe anti-PlGF antibody or ligand.

The PlGF ligands, Mabs, fusion proteins, and fragments thereof also aresuited for the preparation of an assay kit. Such a kit may comprise acarrier means that is compartmentalized to receive in close confinementone or more container means such as vials, tubes and the like, each ofsaid container means comprising the separate elements of theimmunoassay. For example, there may be a container means containing thecapture antibody immobilized on a solid phase support, and a furthercontainer means containing detectably labeled antibodies in solution.Further container means may contain standard solutions comprising serialdilutions of PlGF antigen. The standard solutions of PlGF antigen may beused to prepare a standard curve with the concentration of PlGF antigenplotted on the abscissa and the detection signal on the ordinate. Theresults obtained from a sample containing PlGF antigen may beinterpolated from such a plot to give the concentration of PlGF antigenin the biological sample.

PlGF ligands, anti-PlGF antibodies, fusion proteins, and fragmentsthereof may also be used to detect the presence of the PlGF antigen intissue sections prepared from a histological specimen. Such in situdetection can be used to determine the presence of the PlGF antigen andto determine the distribution of the PlGF antigen in the examinedtissue. In situ detection can be accomplished by applying adetectably-labeled PlGF ligand or antibody to frozen orparaffin-embedded tissue sections. General techniques of in situdetection are well-known to those of ordinary skill. See, for example,Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIANDEVELOPMENT: A PRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987),and Coligan at pages 5.8.1-5.8.8.

PlGF ligands, anti-PlGF antibodies, fusion proteins, and fragmentsthereof can be detectably labeled with any appropriate marker moiety,for example, a radioisotope, an enzyme, a fluorescent label, a dye, achromagen, a chemiluminescent label, a bioluminescent label or aparamagnetic label. Methods of making and detecting suchdetectably-labeled PlGF antibodies are well-known to those of ordinaryskill in the art, and are described in more detail below.

The marker moiety may be a radioisotope that is detected by such meansas the use of a gamma counter or a beta-scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety refersto a molecule that will generate a signal under predeterminedconditions. Examples of marker moieties include radioisotopes, enzymes,fluorescent labels, chemiluminescent labels, bioluminescent labels andparamagnetic labels. The binding of marker moieties to PlGF antibodiescan be accomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin. Chim.Acta 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih etal., Int'l J. Cancer 46: 1101 (1990).

Nucleic Acid Based In Vitro Diagnosis

In particular embodiments, nucleic acids may be analyzed to determinelevels of PlGF expression, particularly using nucleic acid amplificationmethods. Nucleic acid sequences (mRNA and/or cDNA) to be used as atemplate for amplification may be isolated from cells contained in abiological sample, according to standard methodologies. The nucleic acidmay be fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary cDNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

In one example, the determination of PlGF expression is performed byamplifying (e.g. by PCR) the PlGF mRNA or cDNA sequences and detectingand/or quantifying an amplification product by any methods known in theart, including but not limited to TaqMan assay (Applied Biosystems,Foster City, Calif.), agarose or polyacrylamide gel electrophoresis andethidium bromide staining, hybridization to a microarray comprising aPlGF specific probe, Northern blotting, dot-blotting, slot-blotting,etc.

Various forms of amplification are well known in the art and any suchknown method may be used. Generally, amplification involves the use ofone or more primers that hybridize selectively or specifically to atarget nucleic acid sequence to be amplified.

Primers: The term primer, as defined herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty base pairs in length, but longersequences may be employed. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.Methods of primer design are well-known in the art, based on the designof complementary sequences obtained from standard Watson-Crickbase-pairing (i.e., binding of adenine to thymine or uracil and bindingof guanine to cytosine). Computerized programs for selection and designof amplification primers are available from commercial and/or publicsources well known to the skilled artisan. Particular primer sequencesof use in detecting PlGF expression are known (e.g., Regnault et al.,2003, J. Physiol 550:641-56). The skilled artisan will realize that thespecific sequences disclosed therein are exemplary only and thatalternative primer and/or probe sequences may be used in the practice ofthe claimed methods.

Amplification: A number of template dependent processes are available toamplify the marker sequences present in a given sample. One of thebest-known amplification methods is the polymerase chain reaction(referred to as PCR) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159.

One embodiment of the invention may comprise obtaining a suitable samplefrom an individual and detecting a PlGF messenger RNA. Once the tissuesample is obtained the sample may be prepared for isolation of thenucleic acids by standard techniques (eg, cell isolation, digestion ofouter membranes, Oligo dT isolation of mRNA etc.) The isolation of themRNA may also be performed using kits known to the art (Pierce, APBiotech, etc). A reverse transcriptase PCR amplification procedure maybe performed in order to quantify an amount of mRNA amplified. Methodsof reverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable DNA polymerases.

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the PlGFantigen, such as metastatic cancer.

In Vivo Diagnosis

PlGF ligands and/or antibodies are of use for in vivo diagnosis. Methodsof diagnostic imaging with labeled peptides or MAbs are well-known. Forexample, in the technique of immunoscintigraphy, PlGF ligands orantibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as fluorine-18 ¹⁸F), gallium-68(⁶⁸Ga), and iodine-124 (¹²⁴I). Such imaging can be conducted by directlabeling of the PlGF ligand, or by a pretargeted imaging method, asdescribed in Goldenberg et al, “Antibody Pretargeting Advances CancerRadioimmunodetection and Radiotherapy,” (submitted MS), see also U.S.Patent Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

For diagnostic imaging, radioisotopes may be bound to the PlGF ligand orantibody either directly, or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid. For example, see Shih et al., supra,and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to PlGFantibody and are appropriate for diagnostic imaging include ^(99m)Tc and¹¹¹In.

The PlGF ligands, antibodies, fusion proteins, and fragments thereofalso can be labeled with paramagnetic ions and a variety of radiologicalcontrast agents for purposes of in vivo diagnosis. Contrast agents thatare particularly useful for magnetic resonance imaging comprisegadolinium, manganese, dysprosium, lanthanum, or iron ions. Additionalagents include chromium, copper, cobalt, nickel, rhenium, europium,terbium, holmium, or neodymium. PlGF ligands, antibodies and fragmentsthereof can also be conjugated to ultrasound contrast/enhancing agents.For example, one ultrasound contrast agent is a liposome that comprisesa humanized PlGF IgG or fragment thereof. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In apreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome. Still more preferred, the liposome isgas filled.

Imaging Agents and Radioisotopes

In certain embodiments, the claimed peptides or proteins may be attachedto imaging agents of use for imaging and diagnosis of various diseasedorgans, tissues or cell types. Many appropriate imaging agents are knownin the art, as are methods for their attachment to proteins or peptides(see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporatedherein by reference). Certain attachment methods involve the use of ametal chelate complex employing, for example, an organic chelating agentsuch a DTPA attached to the protein or peptide (U.S. Pat. No.4,472,509). Proteins or peptides also may be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶², copper⁶⁴, copper⁶⁷, ¹⁵²Eu, fluorine¹⁸, gallium⁶⁷, gallium⁶⁸,³hydrogen, iodine¹²³, iodine¹²⁴, iodine¹²⁵, iodine¹³¹, indium¹¹¹,⁵²iron, ⁵⁹iron, ³²phosphorus, ³³phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸,Sc⁴⁷, ⁷⁵selenium, silver¹¹¹, ³⁵sulphur, technicium^(94m)technicium^(99m) yttrium⁸⁶ and yttrium⁹⁰. ¹²⁵I is often being preferredfor use in certain embodiments, and technicium^(99m) and indium¹¹¹ arealso often preferred due to their low energy and suitability for longrange detection.

Radioactively labeled proteins or peptides may be produced according towell-known methods in the art. For instance, they can be iodinated bycontact with sodium or potassium iodide and a chemical oxidizing agentsuch as sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Proteins or peptides may be labeled withtechnetium-^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the peptide. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA,NOTA, porphyrin chelators and ethylene diaminetetracetic acid (EDTA).Also contemplated for use are fluorescent labels, including rhodamine,fluorescein isothiocyanate and renographin.

In certain embodiments, the claimed proteins or peptides may be linkedto a secondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart in light and is described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference. These fluorescent labels arepreferred-for in vitro uses, but may also be of utility in in vivoapplications, particularly endoscopic or intravascular detectionprocedures.

In alternative embodiments, PlGF ligands, antibodies, or other proteinsor peptides may be tagged with a fluorescent marker. Non-limitingexamples of photodetectable labels include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY—FL, BODIPY—R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. These and other luminescent labelsmay be obtained from commercial sources such as Molecular Probes(Eugene, Oreg.).

Chemiluminescent labeling compounds of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester, or a bioluminescent compound such asluciferin, luciferase and aequorin. Diagnostic immunoconjugates may beused, for example, in intraoperative, endoscopic, or intravascular tumoror disease diagnosis.

In various embodiments, labels of use may comprise metal nanoparticles.Methods of preparing nanoparticles are known. (See e.g., U.S. Pat. Nos.6,054,495; 6,127,120; 6,149,868; Lee and Meisel, J. Phys. Chem.86:3391-3395, 1982.) Nanoparticles may also be obtained from commercialsources (e.g., Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc.,Warrington, Pa.). Modified nanoparticles are available commercially,such as Nanogold® nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.).Functionalized nanoparticles of use for conjugation to proteins orpeptides may be commercially obtained.

Cross-Linkers

In some embodiments, proteins or peptides may be labeled using variouscross-linking reagents known in the art, such as homo-bifunctional,hetero-bifunctional and/or photoactivatable cross-linking reagents.Non-limiting examples of such reagents include bisimidates;1,5-difluoro-2,4-(dinitrobenzene); N-hydroxysuccinimide ester of subericacid; disuccinimidyl tartarate; dimethyl-3,3′-dithio-bispropionimidate;N-succinimidyl-3-(2-pyridyldithio)propionate;4-(bromoaminoethyl)-2-nitrophenylazide; and 4-azidoglyoxal. In anexemplary embodiment, a carbodiimide cross-linker, such as DCCD or EDC,may be used to cross-link acidic residues to amino or other groups. Suchreagents may be modified to attach various types of labels, such asfluorescent labels.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes. Homobifunctional reagents that carry two identicalfunctional groups proved to be highly efficient in inducingcross-linking between identical and different macromolecules or subunitsof a macromolecule, and linking of polypeptide ligands to their specificbinding sites. Heterobifunctional reagents contain two differentfunctional groups. By taking advantage of the differential reactivitiesof the two different functional groups, cross-linking can be controlledboth selectively and sequentially. The bifunctional cross-linkingreagents can be divided according to the specificity of their functionalgroups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specificgroups. Of these, reagents directed to free amino groups have becomeespecially popular because of their commercial availability, ease ofsynthesis and the mild reaction conditions under which they can beapplied. A majority of heterobifunctional cross-linking reagentscontains a primary amine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, incorporated herein by reference). The cross-linking reagentscombine a nucleophilic hydrazide residue with an electrophilic maleimideresidue, allowing coupling in one example, of aldehydes to free thiols.The cross-linking reagent can be modified to cross-link variousfunctional groups.

Vectors for Cloning, Gene Transfer and Expression

In certain embodiments, expression vectors may be employed to expresspeptides or proteins, such as fusion proteins, which can then bepurified and used. In other embodiments, the expression vectors may beused, for example, in gene therapy. Expression requires that appropriatesignals be provided in the vectors, and which include various regulatoryelements, such as enhancers/promoters from either viral or mammaliansources that drive expression of the genes of interest in host cells.Elements designed to optimize messenger RNA stability andtranslatability in host cells also are known.

Regulatory Elements

The terms “expression construct” or “expression vector” are meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid codingsequence is capable of being transcribed. In preferred embodiments, thenucleic acid encoding a gene product is under transcriptional control ofa promoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent and under the controlof a promoter that is capable of being expressed in a human cell.Generally speaking, such a promoter might include either a human orviral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphatedehydrogenase promoter can be used to obtain high-level expression ofthe coding sequence of interest. The use of other viral or mammaliancellular or-bacterial phage promoters which are well-known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose.

Where a cDNA insert is employed, typically one will typically include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed, such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression construct is a terminator. These elements can serve toenhance message levels and to minimize read through from the constructinto other sequences.

Selectable Markers

In certain embodiments, the cells containing nucleic acid constructs maybe identified in vitro or in vivo by including a marker in theexpression construct. Such markers would confer an identifiable changeto the cell permitting easy identification of cells containing theexpression construct. Usually the inclusion of a drug selection markeraids in cloning and in the selection of transformants. For example,genes that confer resistance to neomycin, puromycin, hygromycin, DHFR,GPT, zeocin, and histidinol are useful selectable markers.Alternatively, enzymes such as herpes simplex virus thymidine kinase(tk) or chloramphenicol acetyltransferase (CAT) may be employed.Immunologic markers also can be employed. The selectable marker employedis not believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable markers are well known to one of skill inthe art.

Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments, the expression construct comprises avirus or engineered construct derived from a viral genome. The abilityof certain viruses to enter cells via receptor-mediated endocytosis, tointegrate into host cell genome, and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (Ridgeway, In: Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, Rodriguez et al., eds.,Stoneham: Butterworth, pp. 467-492, 1988; Nicolas and Rubenstein, In.Vectors. A survey of molecular cloning vectors and their uses, Rodriguezand Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Baichwaland Sugden, 1986, In: Gene Transfer, Kucherlapati R, ed., New York,Plenum Press, pp. 117-148; Temin, In. Gene Transfer, Kucherlapati R,ed., New York, Plenum Press, pp. 149-188, 1986). Preferred gene therapyvectors are generally viral vectors.

Although some viruses that can accept foreign genetic material arelimited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preeparing replication infective viruses are well known in the art.

In using viral delivery systems, one will desire to purify the virionsufficiently to render it essentially free of undesirable contaminants,such as defective interfering viral particles or endotoxins and otherpyrogens such that it will not cause any untoward reactions in the cell,animal or individual receiving the vector construct. A preferred meansof purifying the vector involves the use of buoyant density gradients,such as cesium chloride gradient centrifugation.

DNA viruses used as gene vectors include the papovaviruses (e.g., simianvirus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwaland Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden,1986). One of the preferred methods for in vivo delivery involves theuse of an adenovirus expression vector. Although adenovirus vectors areknown to have a low capacity for integration into genomic DNA, thisfeature is counterbalanced by the high efficiency of gene transferafforded by these vectors. “Adenovirus expression vector” is meant toinclude, but is not limited to, constructs containing adenovirussequences sufficient to (a) support packaging of the construct and (b)to express an antisense or a sense polynucleotide that has been clonedtherein.

Generation and propagation of adenovirus vectors which are replicationdeficient depend on a helper cell line, designated 293, which istransformed from human embryonic kidney cells by Ad5 DNA fragments andconstitutively expresses E1 proteins (Graham et al., J. Gen. Virol.,36:59-72, 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, Cell, 13:181-188, 1978), the current adenovirusvectors, with the help of 293 cells, carry foreign DNA in either the E1,the E3, or both regions (Graham and Prevec, In. Methods in MolecularBiology. Gene Transfer and Expression Protocol, E. J. Murray, ed.,Humana Press, Clifton, N.J., 7:109-128,1991).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As discussed,the preferred helper cell line is 293.

Racher et al. (Biotechnology Techniques, 9:169-174, 1995) disclosedimproved methods for culturing 293 cells and propagating adenovirus. Inone format, natural cell aggregates are grown by inoculating individualcells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)containing 100-200 ml of medium. Following stirring at 40 rpm, the cellviability is estimated with trypan blue. In another format, Fibra-Celmicrocarriers (Bibby Sterlin, Stone, UK) (5 g/l) are employed asfollows. A cell innoculum, resuspended in 5 ml of medium, is added tothe carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary,with occasional agitation, for 1 to 4 h. The medium is then replacedwith 50 ml of fresh medium and shaking is initiated. For virusproduction, cells are allowed to grow to about 80% confluence, afterwhich time the medium is replaced (to 25% of the final volume) andadenovirus added at an MOI of 0.05. Cultures are left stationaryovernight, following which the volume is increased to 100% and shakingis commenced for another 72 hr.

Other gene transfer vectors may be constructed from retroviruses. Theretroviruses are a group of single-stranded RNA viruses characterized byan ability to convert their RNA to double-stranded DNA in infected cellsby a process of reverse-transcription (Coffin, In. Virology, Fields etal., eds., Raven Press, New York, pp. 1437-1500, 1990). The resultingDNA then stably integrates into cellular chromosomes as a provirus anddirects synthesis of viral proteins. The integration results in theretention of the viral gene sequences in the recipient cell and itsdescendants. The retroviral genome contains three genes, gag, pol, andenv. that code for capsid proteins, polymerase enzyme, and envelopecomponents, respectively. A sequence found upstream from the gag genecontains a signal for packaging of the genome into virions. Two longterminal repeat (LTR) sequences are present at the 5′ and 3′ ends of theviral genome. These contain strong promoter and enhancer sequences, andalso are required for integration in the host cell genome (Coffin,1990).

In order to construct a retroviral vector, a nucleic acid encodingprotein of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes, but without the LTR andpackaging components, is constructed (Mann et al., Cell, 33:153-159,1983). When a recombinant plasmid containing a cDNA, together with theretroviral LTR and packaging sequences is introduced into this cell line(by calcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are capableof infecting a broad variety of cell types. However, integration andstable expression require the division of host cells (Paskind et al.,Virology, 67:242-248, 1975).

Other viral vectors may be employed as expression constructs. Vectorsderived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwaland Sugden, 1986; Coupar et al., Gene, 68:1-10, 1988), adeno-associatedvirus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat andMuzycska, Proc. Natl. Acad. Sci. USA, 81:6466-6470, 1984), and herpesviruses may be employed. They offer several attractive features forvarious mammalian cells (Friedmann, Science, 244:1275-1281, 1989;Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., Gene, 68:1-10,1988; Horwich et al., J. Virol., 64:642-650, 1990).

Pharmaceutical Compositions

In some embodiments, a PlGF ligand and/or one or more other therapeuticagents may be administered to a subject, such as a subject with cancer.Such agents may be administered in the form of pharmaceuticalcompositions. Generally, this will entail preparing compositions thatare essentially free of impurities that could be harmful to humans oranimals.

One generally will employ appropriate salts and buffers to rendertherapeutic agents stable and allow for uptake by target cells. Aqueouscompositions may comprise an effective amount of a PlGF binding proteinor peptide, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the PlGF ligands disclosed herein, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The methods and compositions claimed herein may include classicpharmaceutical preparations. Administration of these compositions mayoccur via any common route so long as the target tissue is available viathat route. This includes oral, nasal, buccal, rectal, vaginal ortopical. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal,intraarterial or intravenous injection. Such compositions normally wouldbe administered as pharmaceutically acceptable compositions.

The pharmaceutical forms suitable for use include sterile aqueoussolutions or dispersions and sterile powders for the preparation ofsterile solutions or dispersions. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it is preferable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of thecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

One skilled in the art would know that a pharmaceutical composition canbe administered to a subject by various routes including, for example,orally or parenterally, such as intravenously. In some cases, a PlGFligand may be displayed on the surface of or incorporated into aliposome. Liposomes consist of phospholipids or other lipids, and aregenerally nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

In certain embodiments, an effective amount of a therapeutic agent, suchas a PlGF ligand, must be administered to the subject. An “effectiveamount” is the amount of the agent that produces a desired effect. Aneffective amount will depend, for example, on the efficacy of the agentand on the intended effect. For example, a lesser amount of anantiangiogenic agent may be required for treatment of a hyperplasticcondition, such as macular degeneration or endometriosis, compared tothe amount required for cancer therapy in order to reduce or eliminate asolid tumor, or to prevent or reduce its metastasizing. An effectiveamount of a particular agent for a specific purpose can be determinedusing methods well known to those in the art.

Therapeutic Agents

Chemotherapeutic Agents

In certain embodiments, chemotherapeutic agents may co-administered withone or more anti-angiogenic agents, such as PlGF ligands.Chemotherapeutic agents include, but are not limited to, 5-fluorouracil,bleomycin, busulfan, camptothecin,: carboplatin, chlorambucil, cisplatin(CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin,estrogen receptor binding agents, etoposide (VP16), farnesyl-proteintransferase inhibitors, gemcitabine, ifosfamide, mechlorethamine,melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine,raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC),transplatinum, vinblastine and methotrexate, vincristine, or any analogor derivative variant of the foregoing.

Chemotherapeutic agents and methods of administration, dosages, etc. arewell known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and in “Remington's Pharmaceutical Sciences”,incorporated herein by reference in relevant parts). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.

Hormones

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones. Progestins such as hydroxyprogesteronecaproate, medroxyprogesterone acetate, and megestrol acetate have beenused in cancers of the endometrium and breast. Estrogens such asdiethylstilbestrol and ethinyl estradiol have been used in cancers suchas breast and prostate. Antiestrogens such as tamoxifen have been usedin cancers such as breast. Androgens such as testosterone propionate andfluoxymesterone have also been used in treating breast cancer.

Angiogenic Inhibitors

In certain embodiments, the PlGF ligands disclosed herein may beco-administered with one or more other anti-angiogenic agents, such asangiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies,anti-vascular growth factor antibodies, anti-Flk-1 antibodies,anti-Flt-1 antibodies, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

Immunomodulators

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins and hematopoietic factors, such asinterleukins, colony stimulating factors, interferons (e.g.,interferons-α, -β and -γ) and the stem cell growth factor designated “S1factor.” Examples of suitable immunomodulator moieties include IL-2,IL-6, IL-10, IL-12, IL-18, IL-21, interferon-gamma, TNF-alpha, and thelike.

The term “cytokine” is a generic term for proteins or peptides releasedby one cell population which act on another cell as intercellularmediators. As used broadly herein, examples of cytokines includelymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to a site of treatment. Chemokines include, but are notlimited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the termchemokines. Similarly, the terms immunomodulator and cytokine overlap intheir respective members.

Radioisotope Therapy and Radioimmunotherapy

In some embodiments, the peptides and/or proteins disclosed and claimedherein may be of use in radiionuclide therapy or radioimmunotherapymethods (see, e.g., Govindan et al., 2005, Technology in Cancer Research& Treatment, 4:375-91; Sharkey and Goldenberg, 2005, J. Nucl. Med.46:115S-127S; Goldenberg et al. (submitted MS), “Antibody PretargetingAdvances Cancer Radioimmunodetection and Radioimmunotherapy,” eachincorporated herein by reference.) In specific embodiments, PlGF ligandsmay be directly tagged with a radioisotope of use, as discussed below,and administered to a subject. In alternative embodiments,radioisotope(s) may be administered in a pretargeting method asdiscussed above, using a haptenic peptide or PlGF ligand that isradiolabeled and injected after administration of a bispecific antibodythat localizes at the site of elevated PlGF expression in the diseasedtissue.

Radioactive isotopes useful for treating diseased tissue include, butare not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²³³Ra, ²²⁵Ac, ⁵⁹Fe, 75Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, 149Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclide preferably has a decayenergy in the range of 20 to 6,000 keV, preferably in the ranges 60 to200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and4,000-6,000 keV for an alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5 hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to a PlGFligand such as an anti-PlGF antibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).Alternatively, ⁹⁰Y, which emits an energetic beta particle, can becoupled to a peptide, antibody, fusion protein, or fragment thereof,using diethylenetriaminepentaacetic acid (DTPA).

Additional potential radioisotopes include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au,²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh,¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl,²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated PlGF ligand, antibody or antibody fragment. Forexample, the radiosensitizer can be used in combination with aradiolabeled ligand, antibody or antibody fragment. The addition of theradiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled ligand, antibody or antibody fragmentalone. Radiosensitizers are described in D. M. Goldenberg (ed.), CANCERTHERAPY WITH RADIOLABELED ANTIBODIES, CRC Press (1995), which isincorporated herein by reference in its entirety.

The peptide, antibody, antibody fragment, or fusion protein that has aboron addend-loaded carrier for thermal neutron activation therapy willnormally be effected in similar ways. However, it will be advantageousto wait until non-targeted immunoconjugate clears before neutronirradiation is performed. Clearance can be accelerated using an antibodythat binds to the PlGF ligand. See U.S. Pat. No. 4,624,846 for adescription of this general principle. For example, boron addends suchas carboranes, can be attached to PlGF ligand antibodies. Carboranes canbe prepared with carboxyl functions on pendant side chains, as iswell-known in the art. Attachment of carboranes to a carrier, such asaminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier. Theintermediate conjugate is then conjugated to the PlGF antibody. Afteradministration of the PlGF antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by alpha-emission to produce highly toxic, short-rangeeffects.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one PlGF ligand. Optionally, other kit ingredients mayinclude one or more other anti-angiogenic agents, chemotherapeuticagents, bi-specific antibodies or other ingredients as discussed above.

If the composition containing components for administration is notformulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore separate containers. In some embodiments, the containers may bevials that contain sterile, lyophilized formulations of a compositionthat are suitable for reconstitution. A kit may also contain one or morebuffers suitable for reconsititution and/or dilution of other reagents.Other containers that may be used include, but are not limited to, apouch, tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered to function well in the practice of the invention,and thus can be considered to constitute preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Effects of PlGF on Tumor Cell Growth, Mobility, Angiogenesisand Metastasis

Methods and Materials

Immunohistochemistry, Histopathology and Flow Cytometry

Flow cytometry was performed by standard methods using 1-5 μg/ml primaryantibody, and 1:500 dilution of FITC-labeled secondary antibody(Biosource International, Camarillo, Calif.). Data were collected on aBD FACSCalibur flow cytometer (BD Biosciences) using Cell Questsoftware.

Paraffin-embedded primary breast cancer tissue arrays from US BiomaxInc. (Rockville, Md.) and TARP, NCI (Bethesda, Md.) were stained usingstandard immunohistochemistry procedures (Taylor et al., 2002a) for PlGFand VEGF expression. Tissue arrays were also probed for Flt-1 or NRP-1(ABXIS, Seoul, Korea). Slides were deparaffinized, blocked withappropriate normal serum, and incubated with antibodies purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, Calif.). After incubationwith the primary antibody, biotinylated secondary antibody was applied,followed by quenching of endogenous peroxidase in 3% H₂O₂.avidin-biotin-horseradish peroxidase (HRP) conjugate was applied towashed slides. Slides were incubated 10 min with HRP substrate,3,3′-diaminobenzidine tetrahydro-chloride (DAB, from Sigma, St. Louis,Mo.), and counterstained with hematoxylin (Sigma). Stained slides wereexamined at 100× and rated for intensity of staining by assigning arelative value that reflected the intensity of staining: faint(0.25-0.5), moderate (0.5-1.0), heavy (1.0-2.0), and intense (>2.0);scoring was in 0.25-point increments. All slides were read (blinded) byone researcher, and spot-checked by another.

Only viable-appearing areas of the tumors were assessed, and a valuerepresenting the overall intensity and % of cells that were stained wasassigned to each slide. Staining of extracellular, connective tissue,and white blood cells was not included in the scores. Backgroundstaining (indicated by control Ag8 antibody) was also assessed and thensubtracted. Tumor specimens were considered positive if stainingintensity was o.5 or grater. Human tumor xenograft samples were stainedwith hemotoxylin and eosin.

Cell Lines

To further characterize the cell lines used, MCF-7, MDA-MB-23 1 and -468(American Type Culture collection, Mannassas, Va.) were assayed for PlGFor VEGF expression by flow cytometry. MDA-MB-23 1 and -468, and MCF-7were also tested for expression of the estradiol receptor alpha (SantaCruz Biotechnology) by IHC of cell monolayers. MCF-7 was positive, andMDA-MB-231 and -468 were negative for the estrogen receptor. MDA-MB-231and -468 xenograft tumors were also probed for Flt-1 by IHC, and werepositive. No MCF-7 tumors were available for Flt-1 testing.

Isolation and Selection of Phage

A phage library (Ph.D.-12 Phage Display Peptide Library Kit, New EnglandBiolabs, Inc., Ipswich, Mass.) was panned on recombinant human PlGF-2(PlGF) or recombinant human VEGF 165 (VEGF) (R&D Systems, Flanders,N.J.), according to the supplier's methods. Subsequent panning wasperformed using either PlGF or a peptide corresponding to the putativereceptor-binding site on the PlGF molecule. Phage were submitted tothree rounds of panning, and plated out on agar in a series of 10-folddilutions for titers. Well-isolated plaques were picked from the titerplates and amplified for further investigation.

Sequencing

DNA was isolated from amplified plaques, and sequenced using the primersuggested by the Ph.D. Kit (M13 phage-specific), Thermo SequenaseRadiolabeled Terminator Cycle Sequencing Kit (USB, Swampscott, Mass.).Binding Peptide 1 (BP1) contained 20 amino acids. A control peptide,CPA, was derived by substituting A for the H, R and D residues in BP1.BP1 and CPA were synthesized commercially (University of Georgia) andinvestigated in vitro and in vivo.

Peptide sequences were examined for homology to the putativeligand-binding sites on Flt-1 or PlGF. One peptide, Binding Peptide 1(BP1), had a minor positional homology to the Flt-1 binding site indomain 2, corresponding to Y199 and L204 (Davis-Smyth et al., 1998, JBiol Chem 273:3216-3222; Iyer et al., 2001, J Biol Chem 276:12153-12161,and its sequence was longer than the others. The other peptides were9-12 amino acids in length, and were selected based on consistency ofsequence between pannings. Two plaque sequences from separate phage wereidentical (Binding Peptide 3, BP3).

Sequence of peptides: !Peptide Name? Sequence BP1 SHRYRLAIQLHASDSSSSCV(SEQ ID NO:1) BP2 QDDHLTTGR (SEQ ID NO:2) BP3 QEAFNRLTSRMH (SEQ ID NO:3)BP4 RMPYSEHSAPLG (SEQ ID NO:4)

Testing of Phage or Free Peptide for Binding to PlGF, VEGF or Flt-1

The protocol suggested by the supplier of the phage display system forpanning was used. For each coated well, another well was incubated inbuffer (as a buffer control). After removal of the coating solution,plates were blocked in 2% BSA in PBS for 1.5 h at RT. Another uncoatedplate was also blocked, and used for diluting the phage. After blocking,amplified phage (˜10⁸ phage/ml) were diluted 1:20 in blocking buffer and50 μl of each dilution was added to the PlGF-coated/blocked andblocked-only wells. Phage were allowed to bind at RT for 1-2 h. Plateswere emptied, and washed by dipping and emptying 3× (wash: 0.05%Tween-20 in PBS). Anti-M13-phage antibody (Amersham Biosciences,Buckinghamshire, England), conjugated to HRP, diluted 1:1000 in blockingbuffer, was added to all wells and incubated 1 h at RT. Plates werewashed by dipping, as described. The substrate was added, and after30-60 min, absorbance was read at 410 mn.

Free peptide binding was determined by coating 96-well plates werecoated with 10 μg/ml PlGF, 10 μg/ml recombinant human Flt-1 fusionprotein (R & D Systems), or 10 μg/ml VEGF. Competition assays were donewith 2.5 or 25 U/ml heparin (Sigma, St. Louis, Mo.). BP1, 3, 4, and ascrambled version of BP3 (scrBP3) were synthesized with a linkerfollowed by the FLAG epitope (DYKDDDDK SEQ ID NO:5) at the C-terminus.Thirty minutes after addition of the first reagent, the second reagentwas added, followed by another 30 minute incubation. Peptide binding wasassessed by probing for the FLAG epitope (Prickett et al., Biotechniques7:580-9, 1989; Castrucci et al., J. Virol. 66:4647-53, 1992). Absorbancewas read at 490 nm. In ELISA-based binding assays, CPA bound PlGF andVEGF, but showed attenuated affinity (50%) for Flt-1. The CPA peptidewas consistently inactive in vitro and in vivo.

Tumor Cell Viability

MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, Sigma)assays were performed under conditions of low serum (1%) to determinePlGF effects on cell viability, the toxicity of the peptides, and theireffect on PlGF-mediated breast tumor cell growth (Mosmann, 1983, JImmunol Meth 65:55-63; Modrak et al., 2000, Biochem Biophys Res Commun268:603-606). Peptide concentration was 1-2 μM, and the concentration ofexogenous PlGF was 2 nM. The human breast tumor lines tested were:MDA-MB-231, MDA-MB-468 and MCF-7 (ATCC, Mannasses, Va.). Briefly,96-well plates were seeded with 5-10×10⁴ cells/ml in medium containing1% fetal bovine serum (FBS). After 24 h, cells were treated withindividual peptides at a concentration of 1-2 μM (4 binding peptides),in quadruplicate, and/or rhuPlGF at a 2-nM concentration. After thedesignated time points, 0.5 mg/ml MTT (5 mg/ml stock diluted 1:10 inRPMI medium with no serum) was added to the wells (Mosmann, 1983). Whenpurple crystals were clearly visible in the cells, and the plateappeared to be fully developed, the reaction was terminated with acidalcohol (0.04M HCl in isopropanol). Absorbance of the dissolved crystalswas read at 570 nm. The 24-h time point was chosen because the in vitroeffects of PlGF on tumor cell migration were measurable at this time(see wound-healing assay below). These assays were also performed usingmedium with 10% FBS, with similar results.

Wound-Healing Assay

To determine the functional effects of PlGF, VEGF and BP peptides onbreast tumor and normal human endothelial cells (HECs), wound-healingassays were performed (Verma et al., 2001, Cell Microbiol 3:169-180;Sung et al., 2003, Exp Cell Res 287:209-218; Itokawa et al., Mol. CancerTher. 1:295-302, 2002). MCF-7, MDA-MB-23 1, and MDA-MB-468 breast tumorcell lines or HECs were seeded onto sterile glass coverslips in Petridishes or six-well plates. When the cells were 70-80% confluent, themonolayers were scratched with a sterile plastic pipet tip. Thecoverslips were washed with sterile PBS, and then treated alone or incombinations with PlGF or VEGF (2 nM), peptide (2 μM), or with antibody(0.67 to 1 μg/ml).

Inhibitor concentrations were 50 μM PD98059 (PD98) (mitogen activatedprotein kinase-kinase 1 [MEK1] inhibitor), 5 nM Wortmannin (phosphatidylinositol 3 kinase [PI3K] inhibitor), or human recombinant soluble Flt-1(sFlt-1) (R&D Systems). Peptides or sFlt-1 were incubated with PlGF—,VEGF-containing medium for 10 min before addition to cells. Cells werepre-incubated with PD98 and Wortmannin for 15-30 min before addition ofPlGF. Stained (Wright-Giemsa) and mounted coverslips were examinedmicroscopically. Five to ten 100× fields were evaluated by counting thenumber of cells separated from the wound edges and which appeared to bemigrating toward the center of the wound. Results are reported as theaverage number of cells migrating into the wound/100× field. In somecases data from separate experiments were combined to accommodateinclusion of various treatments in the tables.

Cell Invasion Assays

Cellular invasion was determined by addition of 5×10⁴cells to growthfactor-reduced Matrigel invasion chambers (BD Biosciences DiscoveryLabware, Bedford, Mass.) (Taylor et al., 2003b; Passaniti et al., 1992,Lab Invest 67, 519-528). Additives to the Matrigel® plugs werepre-incubated together in a total volume of 100 μl PBS at RT for 10 min,after which each additive was added to the Matrigel. Growth factors (2.0or 0.2 nM), peptide (2 μg/ml), or antibody (1 μg/ml) were added to cellsin low serum (0.1% fetal bovine serum [FBS]) medium. Baseline andchemoattractant-based invasion (10% FBS) were included. The number ofinvading cells was determined by counting the number of cells on thebottom of the invasion chamber membrane after staining at 100×magnification after 24 h (MDA-MB-231) or 48 h (MCF-7, MDA-MB-468). Theresults of three to five separate experiments are presented.

Effect of Peptide Treatment on Tumor Growth In Vivo

The tissue-culture-grown human breast tumor cell line, MDA-MB-231, wasimplanted subcutaneously (s.c.) on the flanks of nude mice. When tumorswere visible, the mice were weighed and the tumors measured. Treatmentwith peptides was begun when tumors averaged about 0.06-0.12 cm³ involume. Mice were treated with 200-μg peptide in PBS by i.p. injectionat 3-4-day intervals for 4 weeks. Animals were weighed and the tumorsmeasured at each treatment. In a second experiment, the control peptide,CPA, was included. Three days after the final treatment, tumors andlungs were harvested. Tumor burden of the lungs (from experiment 1) wasdetermined by counting colonies (<20 cells), clusters (>20-100 cells),and nodules (>100 cells) seen in all the lung tissue on a total of 4slides or sections for each mouse. Data are presented as the averagenumber of each size of metastasis for each treatment group (3-4mice/group). The total number of each was summed for each treatmentgroup, and divided by the total number of mice in that group. Thepercent inhibition for metastasis was determined by the formula: %inhibition=(# mock-treated−# treated/# mock-treated)×100.

MDA-MB-231, grown in tissue culture, was also implanted in the mammaryfat pad (mfp) of SCID mice (3×10⁶ cells, 4-5 mice/group). In this model,large pulmonary metastases are less likely to develop (Richert et al.,Breast Cancer Res. 7:R819-27, 2005). Peptide treatment was commenced onday 5 after implantation, when small tumors were palpable in all mice (8treatments). Three days after the final peptide treatment, tumors wereremoved and weighed. Lungs were harvested and examined microscopicallyfor metastases, as described above.

Statistical Analyses

Binding, viability, motility, angiogenesis, tumor growth, and metastatictumor burden were evaluated by ANOVA. P<0.05 was considered assignificant.

Results

PlGF is Constitutively Expressed in Primary Human Breast Cancer andBreast Tumor Cell Lines

The frequency of constitutive PlGF and PlGF-receptor expression byprimary breast cancer and breast tumor cell lines was investigated byimmunohistochemical staining of tissue arrays, as discussed above. Theresults, shown in Table 1, demonstrated a higher proportion ofPlGF-positive samples than VEGF (43%-60%, PlGF; 13%-14%, VEGF).Expression of the PlGF receptor, NRP-1, was limited to breast cancerparenchyma (27%), but not tumor endothelium. On the other hand, bothtumor parenchyma and endothelium were positive for Flt-1 (65% and 56%,respectively). TABLE 1 Expression of PlGF, VEGF, NRP-1, and Flt-1 byprimary breast cancer. Marker Array 1 (%) Array 2 (%) PlGF 30/69 (43%)30/50 (60%) VEGF  9/70 (13%)  7/50 (14%) NRP-1 ND 25/94 (27%)Flt-1-tumor ND 31/48 (65%) Flt-1-vessels ND 27/48 (56%)# of moderate to strong staining tumors/total # samples/array. Array 1,TARP. Array 2, commercial source. ND, not done

Lung carcinomas and brain tumors (glioblastomas) were also among thosewith relatively high frequency of expression (32% and 20%, respectively,data not shown). Colonic carcinomas were among the lowest constitutiveexpressers (8%), as were the colonic tumor cell lines (1/4) (not shown).

Human breast cancer cell lines, MCF-7, MDA-MB-23-1, and MDA-MB-468, wereanalyzed for PlGF and VEGF expression by flow cytometry. The percent ofPlGF-positive cells was 29%, 49%, and 38% for MCF-7, MDA-MB-231, andMDA-MB-468, respectively. The percent of VEGF-positive cells was 8%,18%, and 13% for MCF-7, MDA-MB-231, and MDA-MB-468, respectively. Thus,these cell lines were relatively high expressers of PlGF and lowexpressers of VEGF. Both MDA-MB-231 and -468 xenograft tumors expressthe PlGF receptor, Flt-1. MDA-MB-468 stained faintly for NRP-1 (notshown), and NRP-1 expression by MDA-MB-231 has already been documented(Soker et al., J. Biol. Chem. 271:5761-7, 1996). These cell lines wereused for subsequent investigation.

Derivation of PlGF-Binding Peptides

The relatively high frequency of PlGF, NRP-1, and Flt-1 expression bybreast cancer cells suggested that PlGF may have a direct effect onthese cells. To investigate the effects of PlGF on breast cancer cells,PlGF— and Flt-1-binding peptides (BPs) were obtained by panning of aphage peptide library. Binding peptides were obtained after at least 3rounds of panning. Binding peptides 1 and 2 (BP1, BP2) were derived frompanning on rhuPlGF, while binding peptides 3 and 4 (BP3, BP4) werederived from phage first panned for two rounds on a peptidecorresponding to sequences of the putative receptor-binding site ofPlGF, and then on rhuPlGF for the third round. BP3 represents sequencescommon to two different phage plaques.

After isolation, phage were subjected to an ELISA-based binding assay onPlGF-coated plates. The absorbance readings (410 nm) for the phage BP1,was 0.024 (Phage library background 0.003). The results indicated thatbinding of the PlGF-panned phage to PlGF-coated wells was 10-20-foldincreased over background

Peptide BP1 (SHRYRLAIQLHASDSSSSCV SEQ ID NO: I), was synthesized with aC-terminal FLAG epitope, and tested for binding to PlGF and Flt-1, andto VEGF. BP1 bound to PlGF (A490 0.100±0.058) and VEGF165 (A4900.299±0.174), but most strongly to Flt-1 (A490, 0.886±0.096).Flt-1-binding was further tested by addition of unbound Flt-1 to bindingassays where Flt-1 was immobilized. Addition of free Flt-1 (2 nM) causeda 38% decrease in binding of BP1 to immobilized Flt-1 (A490 for 5 μM BP1only, 0.527±0.025; with added free Flt-1 and BP1, 0.327±0.127).

Additional assays were performed to determine if the presence of PlGFwould interfere with the BP1-Flt-1 interaction. PlGF at concentrationsbetween 1 and 100 nM had no significant effect on the binding of BP1 toFlt-1. These findings suggested that BP1 interacted with a site on Flt-1other than domain 2, where the major interactions of PlGF with Flt-1 arelocated. Subsequently, the association of BP1 with the heparin-bindingsites present on both Flt-1 and PlGF-2 was investigated. As shown inFIG. 1, addition of heparin to Flt-1-coated plates before addition ofBP1 resulted in a 64% decrease in BP1 binding (P<0.0002). However, ifBP1 was added prior to addition of heparin, the binding of BP1 wasinhibited by only 13% (P>0.05). Thus, BP1 most likely binds to Flt-1 ator near the heparin-binding site.

BP1 may itself contain a heparin binding motif, as the first sixresidues have the same pattern (XBBXBX) as the first six residues of theheparin binding domain found in vitronectin. This domain may serve asthe core of alternative binding peptide sequences that could exhibitincreased potency with respect to inhibition of angiogenesis, tumormetastasis and/or tumor cell mobility. The skilled artisan will realizethat, for example, randomization of amino acids in the locationsindicated as “X” may potentially result in the production of usefulanalogs of BP1. While the charge distribution may be of significance,substitution of other basic residues (e.g., histidine, arginine, lysine)in the positions indicated as “B” may also result in the production ofuseful analogs of BP1.

No measurable binding of BP1 to VEGF was detected. Unexpectedly, BP1also bound to Flt-1 at 10-fold over background. Specificity of the Flt-1binding was tested by addition of unbound Flt-1 (2 nM) to ELISA-basedbinding assays, and this caused a 38% decrease in binding of 5 μM BP1 toimmobilized Flt-1.

Peptides BP3 and BP4, and a scrambled version of BP3 (BP3scr), also wereassayed as free peptides for binding to PlGF and Flt-1, and did not binddetectably to PlGF at concentrations up to 50 μM. No measurable bindingto Flt-1 was detected for these peptides.

Viability of Breast Tumor Cells when Exposed to PlGF or Peptides

PlGF and all 4 peptides were assayed for their effects on breast tumorcell viability. The cell lines, MCF-7, MDA-MB-231, and MDA-MB-468, werechosen because they typify many primary and metastatic breast cancers.MCF-7 is estrogen receptor-positive and has a moderatelywell-differentiated histology. MDA-MB-231, originally obtained from ametastatic pleural effusion (Cailleau et al., 1974, J Natl Cancer Inst53:661-674), is estrogen receptor-negative, and forms poorlydifferentiated grade III adenocarcinoma xenografts. MDA-MB-468 is alsoestrogen receptor-negative, poorly differentiated, and is tumorigenic innude mice. All three tumors constitutively produce PlGF 3-4-foldcompared to the PlGF-negative colonic tumor cell line, HT-29 (by flowcytometry) and express low levels of the PlGF receptor Flt-1 (data notshown). MDA-MB-231 is the highest producer of PlGF, and MDA-MB-468, thelowest.

Addition of PlGF (2 nM) to tumor cell cultures resulted in increasedcell numbers after 24 h of culture (P<0.04 for MDA-MB-231 andMDA-MB-468) (FIG. 4). To determine the effect of the peptides on thePlGF-stimulated viability and proliferation, peptides (1-2 μM) werecombined with PlGF (2 nM). The results of addition of the peptides toPlGF-containing MTT assays are shown in FIG. 4. Addition of BP4 toPlGF-containing MCF-7 cultures (FIG. 4A) resulted in a significantreduction in cell viability, as did addition of BP3 to MDA-MB-231 (FIG.3C) (P<0.03). All three peptides decreased the viability ofPlGF-containing MDA-MB-468 cultures (FIG. 3B) (P<0.03).

Incubation of tumor cells for 24 h with the peptides alone (2 μM) had nosignificant effect on cell viability, except for MCF-7 incubated withBP1, where the viability was lowered by 21% (P<0.005). After 48 h ofincubation with BP3, MCF-7 also lost viability compared to untreatedcontrols (P>0.05)

PlGF Stimulates Tumor Cell Migration and is Involved in Metastasis

It was determined whether PlGF could increase the likelihood of tumorrecurrence or metastasis. Because metastatic tumor cells displayincreased motility and invasive potential, exogenous PlGF was added tomotility and invasion assays containing the breast cancer cell lines,MCF-7, MDA-MB-231, and MDA-MB-468. PlGF caused a 1.8-2.1-fold increasein motility of MDA-MB-231 and MCF-7 (P<0.01), but did not significantlyaffect the motility of MDA-MB-468. Addition of recombinant soluble Flt-1(2 nM) significantly blocked the stimulatory effect of PlGF by 55% and67% for MDA-MB-231 and MCF-7, respectively (P<0.02) (Table 2). Anti-PlGFantibody (1 μg/ml) also blocked the PlGF-mediated motility of MDA-MB-231and MCF-7 (P<0.02) (Table 2). BP1 also inhibited PlGF-stimulatedmotility in MDA-MB-231 and MCF-7 significantly (P<0.02). Controlpeptide, CPA, had no effect, nor did VEGF (2 nM) (Table 2). Anti-VEGFantibody added to PlGF-containing assays did not affect PlGF-stimulatedmigration of MDA-MB-231, suggesting that the increased motility observedin this cell line was not due to VEGF activity (Table 2). TABLE 2PlGF-stimulated tumor cell motility inhibition by peptide BP1, Flt-1,anti-PlGF antibody, anti-VEGF antibody, and PI3K/MEK1 inhibitors. Growthfactor Relative Cell line (2 nM) Test agent Migrating cells activityMDA-MB-231 0 0 23.8 ± 6.5 PlGF 0 42.0 ± 13.3* 100 PlGF Flt-1 19.1 ± 8.0†45 PlGF BP1 13.6 ± 6.1† 32 PlGF CPA 39.8 ± 10.6 106 PlGF anti-PlGF 17.0± 6† 40 PlGF anti-VEGF 34.4 ± 12.6 82 PlGF PD98059 18.4 ± 8.0‡ 43 PlGFWortmannin 16.4 ± 5.8‡ 39 VEGF 0 11.1 ± 1.8 100 VEGF BP1 14.7 ± 2.1 132VEGF CPA 13.4 ± 4.5 121 VEGF anti-VEGF 11.5 ± 2.3 104 VEGF anti-PlGF13.8 ± 14.6 124 MDA-MB-468 0 0 13.4 ± 9.1 PlGF 0 14.7 ± 9.8 100 PlGFFlt-1  7.8 ± 5.9† 53 PlGF BP1 14.3 ± 8.1 97 PlGF CPA 21.6 ± 6.1 147 PlGFanti-PlGF  7.6 ± 5.7† 52 VEGF 0 14.0 ± 11.3 100 VEGF BP1 15.8 ± 9.5 113VEGF CPA 14.6 ± 8.0 104 VEGF anti-VEGF 14.2 ± 11.5 101 MCF-7 0 0 12.2 ±11.4 PlGF 0 22.1 ± 13.4* 100 PlGF Flt-1 10.4 ± 8.2† 47 PlGF BP1 10.2 ±7.8† 45 PlGF anti-PlGF 10.9 ± 4.9† 49 VEGF 0 12.7 ± 4.1 100 VEGF BP112.9 ± 2.9 102 VEGF CPA 19.4 ± 8.9 111 VEGF anti-VEGF 12.9 ± 4.3 102PlGF, VEGF and Flt-1, 2 nM; peptide, 2 μM. Antibody concentration was 1μg/ml. MEK1 inhibitor, PD98059 (PD98), 50 μM. PI3K inhibitor,Wortmannin, 5 nM. Results are presented as average number of cellsmigrating into the ‘wound’±SD from 5-10-100× microscopicfields/treatment/experiment at 18-24 h. n=2-4 experiments. Values forantibody and inhibitor treatments were from separate experiments whichincluded all relevant controls. In two experiments all values wereapproximately one half of those in the majority of previous experiments,and so they were normalized by a factor of 2, and then averaged withother experiments. UT denotes untreated controls. *P<0.01 (ANOVA)compared to UT; † P<0.02 (ANOVA) compared to PlGF only. ‡ P<0.001(ANOVA) compared to PlGF only. Relative activity was determined bydividing the average value for the treatments by the PlGF— or VEGF-onlyvalue.

Both PI3K and MEK1 activation are often linked to tyrosine kinasereceptor (RTK) intracellular signaling. In addition, active PI3K andMEK1 have been implicated in tumor cell migration (Zeng et al., J. Biol.Chem. 276:26969-79, 2001; Hollande et al., Am. J. Physiol. Gastrointest.Liver Physiol. 280:G910-21, 2001). To investigate whetherPlGF-stimulated motility was due to activation of either of thesekinases, MDA-MB-231 was treated with PlGF and inhibitors of PI3K(Wortmannin, 5 nM) or MEK1 (PD98059, 50 μM [PD98]). PD98 and Wortmanninsignificantly inhibited PlGF-stimulated migration by 68% and 72%,respectively (P<0.001) (Table 2). These results suggest that PlGFstimulates cellular motility through the activation of the PI3K and MEK1pathways, possibly through the activation of PI3K by Flt-1.

Because the ability to invade the basement membrane is essential formetastasis, invasion assays with added PlGF or VEGF were performed.Addition of PlGF at both 2.0 and 0.2 nM resulted in nearly 3-foldincreased invasion of MDA-MB-231 after 24 h (11±8.1 untreated vs.30±13.7 (2.0 nM) or 28±12 (0.2 nM) PlGF-treated, P<0.05) (FIG. 2). VEGF(2.0 nM or 0.2 nM) did not alter the invasion capacity of MDA-MB-231(10±8.0 cells). Addition of peptide BP1 resulted in a 50% decrease inPlGF-stimulated invasion (15±4.2 cells) (P<0.02 vs. PlGF-only). Thecontrol peptide, CPA, did not inhibit the activity of PlGF (35±17.3cells). Addition of anti-PlGF antibody (0.6 μg/ml) to the 0.2 nM PlGFassays resulted in a 46% reduction in invasive cells (15±2.3 cells, oneexperiment). Taken together, these results suggest that PlGF stimulatesinvasiveness in the aggressive tumor cell line, MDA-MB-231, and that theinhibitory peptide, BP1, similar to the PlGF antibody, inhibits thisactivity. Similar results were obtained for MDA-MB-468 and MCF-7, butwere not statistically significant (Table 3). TABLE 3 PlGF increasesinvasion potential of breast cancer cell lines. BP1 inhibitsPlGF-mediated invasive potential. Line Untreated PlGF PlGF + BP1 PlGF +CPA BP1 MCF-7 1 2.1 ± 0.7 0.3 ± 0.0 1.0 ± 0.6 0.7 ± 0.4 MDA-MB-231 1 4.0 ± 2.2*  1.3 ± 0.1† 4.9 ± 2.4 1.0 ± 0.6 MDA-MB-468 1 1.8 ± 0.3 0.6 ±0.4 1.2 ± 0.7 1.2 ± 0.3Average fold-change in the number of cells on the membrane after 24 h(MDA-MB-231) or 48 h (MDA-MB-468 and MCF-7) compared to untreatedcontrols ± SD. Concentration of PlGF, 0.2 nM; peptide, 2 μg/ml. n = 2-4experiments/cell line. *P < 0.01 vs. Untreated. †P < 0.02 vs. PlGF-only.

To test for therapeutic activity of the peptide, BP1, mice wereimplanted with human breast cancer cells, MDA-MB-231, which produceslung metastases spontaneously when implanted as a subcutaneous tumor.When the tumors averaged 0.12 cm³ in volume, either mock-treatment ortherapy with BP1 was initiated at 200 μg, two times per week for 4weeks. Mice receiving BP1 showed a 37% reduction in tumor size comparedto mock-treated controls. In a subsequent experiment, treatment wascommenced when tumors were 0.07-0.08 cm³. After 4 weeks of treatment, asdescribed, the mean volume of the mock- and CPA-treated tumors was 0.25and 0.33 cm³, respectively, whereas BP1-treated tumors averaged 0.04 cm³(P<0.05 BP1 vs. mock- or CPA-treated tumors) (FIG. 3A).

Three days after treatment, primary tumors and lungs were removed formicroscopic examination. Typically, the lungs of mock-treated animalscontained multiple large and small metastases, with many foci of tumorgrowth mostly surrounding the lung blood vessels, with spread into thesurrounding parenchyma (FIG. 3B). The lungs of mock-treated controlsaveraged 78±25 nodules, 76±19 clusters, and 36±6 colonies/total lungtissue/mouse. In contrast, the BP1-treated lungs averaged 5±2, 9±2, and5±2 nodules, clusters, and colonies, respectively, per mouse. Thisconstituted a 94% decrease in the number of metastatic lung nodules inthe peptide-treated animals (P<0.007).

In an orthotopic mammary fat pad (mfp) model using MDA-MB-231, treatmentwith BP1 decreased tumor weight by 23% (mock-treated, 0.423±0.089 g;CPA, 0.416±0.083 g; BP1, 0.345±0.095), which did not reach statisticalsignificance. The mfp-implanted MDA-MB-231 did not produce large lungnodules, possibly due to the short duration of the experiment (4.5weeks). However, micrometastases (20 to >100 cells) were apparentadjacent to pulmonary veins (not shown). Micrometastasis counts were3.4±1.9, 2.8±1.3, and 0.6±0.5 per mouse for mock-treated, CPA, and BP1,respectively (P<0.02 vs. mock or CPA-treated), a reduction in metastasesof 82%.

Mice bearing MDA-MB-468 xenografts (average tumor volume: 0.19 cm³) weretreated with i.p. injections of individual peptides (200 μg/dose) (FIG.5). After 4 weeks of twice-weekly treatment, tumor growth was inhibitedby 49% and 58% in mice treated with BP1 (FIG. 5A) or BP3 (FIG. 5B),respectively. The effect of BP3 on tumor growth was apparent after 2weeks of treatment (FIG. 5B), whereas the inhibitory effect of BP1 wasevident after one week (FIG. 5A). The average changes in individualtumor volumes during treatment with BP1 were significant (P<0.05) atdays 10, 14, 17 and 24 (FIG. 5A). Treatment with BP2 was ineffective;tumors continued to grow at almost the same rate as the mock-treatedcontrols (results not shown). BP4 treatment produced a 20% inhibition oftumor growth (results not shown).

Inhibition of Angiogenesis

To further investigate the effect of PlGF binding peptides onendothelial cell migration and angiogenesis, Matrigel basement membraneplugs containing PlGF, BP1, BP2, or PBS were implanted s.c. on theflanks of nude mice. The number of interior microvessels in the PBScontrols was 16.2±11.4/mm². PlGF-only implants contained 28.9±17.2/mm²(1.8-fold vs. mock-treated implants), and implants with PlGF and BP1contained only 12.3±13.0 microvessels/mm² (P<0.02).

Discussion

Several studies have indicated that PlGF expression by cancers,including breast, correlates with recurrence, metastasis and mortality(Wei et al., Gut 54:666-72, 2005; Chen et al., Cancer Lett. 213:73-82,2004; Parr et al., Eur. J. Cancer 41:2819-27, 2005; Weidner et al., Am.J. Pathol. 143:401-9, 1993; Zhang et al., World J. Surg. Oncol. 3:68,2005). PlGF is also associated with a number of pathological states(Carmeliet et al., Nat. Med. 7:575-83, 2001) and tumorneovascularization (Li et al., FASEB J. 20:1495-97, 2006; Taylor et al.,Int. J. Cancer 1-5:158-69, 2003).

Clinical studies of VEGF and PlGF in human cancer are conflicting. Forinstance, in one report, PlGF, rather than VEGF, stimulated growth ofPhiladelphia chromosome-positive acute myelogenous leukemias ex vivo(Ikai et al., Eur. J. Haematol. 75:273-9, 2005). On the other hand, VEGFwas associated with renal cell cancer stage, histological grade, as wellas its vascularity and venous invasion (Matsumoto et al. Anticancer Res.23:4953-8, 2003). These authors reported that PlGF also was anindependent prognostic factor for this cancer.

Multiple anti-VEGF agents have been developed, and some, includingblocking antibodies, small molecules that prevent VEGF-receptor binding,and anti-sense oligonucleotides, have been tested in clinical trials(Whatmore et al., 2002, Angiogenesis 5:45-51; Mulligan-Kehoe et al.,2002, J Biol Chem 277:49077-49089; Shi and Siemann, 2002, Br J Cancer87:119-126; Yang et al., 2003, NEJM349, 427-434). The results of thesestudies suggest that tumor angiogenesis is complex and redundant; i.e.,that there may be ‘back-up’ mechanisms that establish a blood supply tomalignancies when VEGF is diminished by treatment, or in tumor typeswith low VEGF expression. PlGF is likely to be involved in thisfunctional redundancy. PlGF is also arteriogenic, causing the formationof arteries from pre-existing anastomoses (Pipp et al., 2003).

The data presented above show that PlGF enhances the metastaticpotential of breast cancer cells, because it stimulates motility andinvasiveness, which are associated with the epithelial-to-mesenchymaltransformation that characterizes metastasis in tumors. In contrast,exogenously added VEGF had no effect on tumor cell motility orinvasiveness in these assays. These results differ from those ofBachelder et al. (Cancer Res. 62:7203-6, 2002), where VEGF was found tobe a stimulator of invasion (MDA-MB-231). However, this study did notinclude PlGF; therefore, comparisons of the growth factors cannot bemade. Our results are based on the presence of PlGF or VEGF in the tumorcell environment, whereas Bachelder et al. (2002) used RNAi to suppressVEGF production by the cells, the results of which may differ fromVEGF's paracrine effects.

Another report documented an inhibitory role for PlGF when it isoverexpressed by tumor lines that are normally low-PlGF and high-VEGFexpressers (Xu et al., Cancer Res. 6:3971-7, 2006. This pattern ofexpression differs from that of the breast lines used in the presentstudy, which were high-PlGF, low-VEGF expressers, a pattern that mimicsprimary human breast carcinomas. At least one other study showed thatVEGF and PlGF synergize to stimulate angiogenesis in pathologicalconditions (Carmeliet et al., 2001). The contribution of PlGF or VEGF totumor pathology is most likely complex, and may in part be due to theirrelative abundance in the tumor microenvironment, as well as thepresence of their active receptors on tumor cells and endothelium.

As disclosed herein, the capacity of PlGF to stimulate cancer cellmotility and invasion was inhibited with anti-PlGF antibody, as well aswith BP1, the PlGF-2/Flt-1 antagonistic peptide developed. It could besurmised that if PlGF were inhibitory, then its removal would allowVEGF-mediated activity to proceed, which was not the case.

Potential intracellular mechanisms for the stimulatory effect ofexogenous PlGF were investigated, and the results showed that the PI3Kand MEK1 pathways likely participate in PlGF-stimulated migration. Thesepathways have been linked previously to cell migration (Hollande et al.,2001), but these kinases also participate in other processes thatpromote cancer progression, including translation of proteins, generegulation, proliferation, invasion, and resistance to apoptosis (Zenget al., J. Biol. Chem. 276:26969-79, 2001; Hollaned et al., 2001; Belkaet al., Int. J. Radiat. Oncol. Biol. Phys. 58:542-54, 2004; Pommery etal. Ann. Pharm. Fr. 63:69-75, 2005; Bancroft et al., Clin. Cancer Res.7:435-42, 2001; Mekhail et al., Cell Cycle 3:1027-9, 2004; Pollheimer etal., Angiogenesis 3:159-66, 1999). PlGF-expressing tumor cells mayobtain survival benefits through autocrine/paracrine pathways thatrender them more resistant to death signals and which allow them tomigrate toward a blood supply when needed.

In order to elucidate the role of PlGF in tumor and endothelial cellbiology, we developed an antagonistic peptide, BP1, which inhibited theactivity of PlGF on both tumor and endothelial cells, and affectedspontaneous metastasis in vivo. The results herein indicate that BP1antagonizes PlGF-2/Flt-1-heparin associations. Heparin-binding isessential for the full activation of some receptor tyrosine kinases(Park et al., Bioechem. Biophys. Res. Comm. 264:730-4, 1999;Schlessinger et al. Molec. Cell. 6:743-50, 2000; Ito et la.,Angiogenesis 3:159-66, 1999), which is supported by our observation thatinhibition of PI3K and MEK1 pathways often linked with receptor tyrosinekinases, such as Flt-1, prevented cell migration stimulated by PlGF. Bypreventing the close association of heparin with PlGF-2 and Flt-1, BP1may prevent the transmission of activation signals to the interior ofthe cell.

Crystallization studies of the FGF—FGFR-heparin complex document thatheparin makes multiple contacts with both the ligand and the receptor.This association enhances dimerization of the receptor, which isnecessary for activation (Park and Lee, 1999; Schlessinger et al., 2000;Ito and Claesson-Welsh, 1999, Angiogenesis 3:159-166). By interferingwith this complex, BP1 may prevent this close association of heparinwith PlGF and Flt-1. We have found that exogenous heparin included inangiogenesis assays augments the formation of vessels even in theabsence of added growth factors, such as VEGF or PlGF (unpublisheddata). In this case, the increased angiogenesis is most likely due toheparin-mediated enhancement of interactions between growth factorsendogenous to the Matrigel and receptors on cells migrating into theimplants.

In addition to inhibiting PlGF-mediated migration, BP1 was able toinhibit the growth of microvessels in PlGF-loaded basement membraneimplants. This inhibition of endothelial cell migration and theestablishment of vessels in tumors, taken together with the migrationassay data, suggest that the function of PlGF in breast cancer pathologyis to stimulate endothelial cell migration and, possibly, tube formation(data not shown), and to also cause movement of tumor cells themselvestoward the blood supply.

Bae et al. (Clin. Cancer Res. 11:2651-61, 2005) recently reported theanti-cancer potential of a Flt-1 antagonistic peptide that inhibitedVEGF binding to Flt-1, as well as the growth and metastasis ofVEGF-secreting colon tumor xenografts. The activity of this peptide wasdetermined by its interaction with endothelial cells, using VEGF as thestimulator of migration and proliferation, rather than tumor cells. Itis interesting that the peptide used by these authors also inhibited thebinding of PlGF to Flt-1, and part of its efficacy may stem from thisinteraction. Their peptide, however, appears to differ from BP1, becauseit interferes with the Flt-1 domain-2 growth factor binding site (Bae etal., 2005), with no evidence of interaction with the heparin-bindingdomain. Thus, the peptide presented by Bae et al. most likely functionsdifferently than BP1.

The in vivo role of PlGF in cancer was studied by treating human breastcancer xenografts that spontaneously metastasize with BP1. Treatmentwith the BP1 peptide arrested s.c. MDA-MB-231 tumor growth and decreasedthe occurrence of pulmonary metastases by 94%, and by 82% in anorthotopic (mfp) model. These results demonstrate the growth- andmetastasis-inhibiting properties of anti-Flt-1 peptides, such as BP1,indicating that this receptor may play a key role in the progression ofcertain cancers, and represents a target for anticancer therapy.

In summary, we have demonstrated increased expression of PlGF in someprimary human cancers and cell lines, especially breast cancer. PlGFincreased the proliferation of breast cancer cell lines, MDA-MB-231 and-468, and the motility of MCF-7 and MDA-MB-231. To our knowledge, thisis the first report documenting the direct activation of malignant cellsby PlGF. Further, we have isolated a peptide, BP1, that binds both PlGFand Flt-1 and which ultimately disrupts the activity of the PlGF-2/Flt-1ligand receptor pair in vitro. The activities blocked by BP1 includePlGF-stimulated tumor cell viability and motility, as well as theformation of microvessels in basement membrane implants. In addition,treatment of tumor-bearing mice with peptide BP1 inhibited the s.c.growth of the breast tumor xenograft, MDA-MB-468, and markedly reducedthe establishment of lung metastases by MDA-MB-231.

Example 2 Inhibition of PlGF Mediated Angiogenesis in a Mouse CornealAssay

The ability of PlGF ligands to block PlGF-mediated angiogenesis incorneal tissues in vivo is investigated. Corneal micropockets arecreated using a modified von Graefe cataract knife in both eyes of male5-6-wk-old C57B16/J mice. Micropellets comprised of a sustained releaseformulation of polyglucuronic acid/polylactic acid containing 100 ng ofPlGF or PlGF with 1 μM BP-1, BP-2, BP-3 or BP-4 are implanted into eachcorneal pocket. Eyes are examined by a slit-lamp biomicroscope on day 5after pellet implantation. Vessel length and neovascularization aremeasured.

PlGF induces a strong angiogenic response with formation of a highdensity of microvessels. Addition of BP-1, BP-3 or BP-4 inhibits PlGFmediated angiogenesis in corneal tissues. Treatment of a subject ofdiabetic retinopathy or macular degeneration results in an inhibition ofblood vessel formation and ameliorates the condition.

Example 3 BP1 Fusion Proteins and Conjugates

For purposes of oral administration or in other embodiments, BP1 and itsanalogs may be recombinantly or chemically linked to a carrier protein.For linking peptides composed of the 20 common L-amino acids found innaturally occurring proteins, recombinant DNA methods are preferred. Forlinking peptide mimetics or peptides that contain D-amino acids,modified amino acids, or unnatural amino acids, only chemicalconjugation methods are currently feasible. As disclosed herein, ageneral method of preparing conjugates of BP1 linked to thecarbohydrates of the Fc of human IgG1 (hFc) is provided. Methods ofconstructing three exemplary fusion proteins comprised of BP1 and hFcare disclosed below.

A vector for expressing hFc in myeloma cells is prepared using standardrecombinant DNA techniques. The expression vector shCD20-Fc-pdHL2 (FIG.6) is used as the DNA template to amplify the coding sequences for Fc(hinge, CH2, and CH3 domains) by PCR. Two PCRs are performed. The firstPCR is to amplify the entire leader peptide sequence (fragment A) usingthe pair of primers shown below. 5′ Xba I: TCTAGAGCACACAGGACCTC (SEQ IDNO:6) 3′ Hind3: GAAGCTTGGAGTGGACACCT (SEQ ID NO:7)

The second PCR is to amplify hinge and CH2 (fragment B) using the pairof primers shown below: 5′ Hind3: AAGCTTCCGACAAAACTCAC (SEQ ID NO:8)3′ Sac2: CCGCGGCTTTGTCTTGGCAT (SEQ ID NO:9)

Following digestion of shCD20-Fc-pdHL2 with XbaI and Sac2, the largerDNA fragment is ligated to fragments A and B to obtain the expressionvector for Fc (hFc-pdHL2).

Example 4 Conjugating BP1 to hFc

Myeloma cells are transfected with hFc-pdHL2 and screened for positiveclones. The highest producer is grown in roller bottles and the culturesupernatant is purified on Protein A to obtain hFc. Conjugation of BP1to hFc is performed using one of the two methods described below. In thefirst method, hFc is oxidized by sodium periodate to generate aldehydegroups in the carbohydrates. Following a desalting step, the oxidizedhFc is derivatized with a hetero-crosslinker such as BMPH (Pierce,Product # 22297) to introduce maleimide groups via the reaction ofhydrazide with aldehyde. Following removal of excess reagents, themaleimide-coupled hFc is conjugated to BP1 via the reaction of thecysteine residue in BP1. An alternative method is to couple BMPH to BP1,isolate the resulting product using reversed-phase HPLC, and reactBMPH—BP1 with oxidized hFc.

Example 5 Expression Vector for BP1-hFc

In alternative embodiments, BP1-hFc is constructed as a fusion proteincomposed of two identical polypeptides, each composed of BP1 linked tothe hinge, CH2 and CH3 of human IgG1 via a flexible linker, such as(GGGGS)₃. The two polypeptides are covalently associated by twodisulfide bonds formed from the cysteine residues in the hinge. BP1-Fcis expected to confer the following advantages: (1) the affinity of BP1for the heparin binding domain of VEGFR1 would be enhanced due tobivalent BP1; (2) the increase in molecular size (˜60 kDa) could betterexclude heparin from binding to VEGFR1, resulting in a more efficientinhibition of the interaction between PlGF and VEGFR1; and (3) thepresence of Fc would prolong the serum half life and enable oral orpulmonary delivery by binding to the neonatal Fc receptor (FcRn)expressed in epithelial cells.

A vector for expressing BP1-Fc in myeloma cells is constructed asfollows. Briefly, three long oligonucleotides are synthesized with 10-15bp overlap to cover the 114 bp sequence encoding BP1 and the (GGGGS)₃linker, with HindIII sites added at the ends. The oligonucleotides areannealed and extended with DNA polymerase. The resulting DNA fragmentcontaining BP1 and the linker is purified and cloned into anintermediate vector, followed by sequencing confirmation. The fragmentis then isolated by digesting the intermediate vector with HindIII, andcloned into HindIII-cut Fc-pdHL2 (see Example 3). Since there are 2possible orientations, the correct one is identified by double enzymedigestion and isolated (BP1-hFc-pdHL2)

Myeloma cells are transfected with BP1-hFc-pdHL2 and selected for highproducing clones. The highest producer is grown in roller bottles andthe culture supernatant is purified on Protein A to obtain BP1-hFc.

Example 6 Dimeric BP1-hFc (BP1-hFc-ddd2)

A fusion protein composed of dimeric BP1-hFc is obtained by appending aspecific peptide sequence termed ddd2 (SEQ ID NO:10) to the C-terminusof BP1-hFc. The dimeric BP1-hFc is stabilized by disulfide bonds andcontains four BP1. ddd2 (SEQ ID NO:10)MCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Example 7 BP1-hFc x anti-VEGFR2 Fab (˜160 kDa)

BP1-Fc is linked by disulfide bond to anti-VEGFR2 Fab using the A2Btechnology to generate a conjugate of ˜160 kDa capable of targeting bothVEGFR1 and VEGFR2. The B-form of anti-VEGFR2 is first generated byappending a specific peptide sequence termed ad2 (SEQ ID NO:11) to theC-terminus of CH1. To generate BP1-hFc x anti-VEGFR1 Fab, BP1-hFc-ddd2(Example 6) is mixed with the B-form of anti-VEGFR2 and reduced withTCEP for 1 h. Following removal of TCEP, DMSO is added to a finalconcentration of 10% to induce the formation of disulfide bonds betweenthe ddd2 and ad2 present in each of the two parental proteins. ad2GGCGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:11)

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A composition comprising a placenta growth factor (PlGF) ligand,wherein the ligand inhibits angiogenesis, tumor growth or tumormetastasis.
 2. The composition of claim 1, wherein the ligand is apeptide.
 3. The composition of claim 2, wherein the peptide isidentified by phage display against PlGF.
 4. The composition of claim 2,wherein the peptide comprises at least 12 contiguous amino acid residuesselected from the sequences of BP-1 (SEQ ID NO:1), BP-2 (SEQ ID NO:2),BP-3 (SEQ ID NO:3) or BP-4 (SEQ ID NO:4).
 5. The composition of claim 4,wherein the peptide comprises the sequence of BP-1 (SEQ ID NO:1).
 6. Thecomposition of claim 1, wherein the ligand comprises an antibody,antibody fragment, chimeric antibody, humanized antibody, humanantibody, human antibody fragment or antibody analog.
 7. The compositionof claim 2, wherein the peptide is in a linear or a cyclic conformation.8. The composition of claim 7, wherein the peptide comprises a cysteineresidue at each end of the peptide and the cysteine residues are bondedto each other to form a cyclic peptide.
 9. The composition of claim 1,wherein the tumor is a lymphoma, leukemia, myeloma, sarcoma, glioma,melanoma, or carcinoma.
 10. The composition of claim 9, wherein thetumor is a breast cancer.
 11. The composition of claim 2, wherein thepeptide binds to both PlGF and Flt-1 (Fms-like tyrosine kinasereceptor).
 12. The composition of claim 11, wherein the peptide binds tothe heparin binding sites on PlGF-2 and Flt-1.
 13. The composition ofclaim 12, wherein the peptide inhibits heparin binding to PlGF and/orFlt-1.
 14. The composition of claim 12, wherein heparin inhibits bindingof the peptide to PlGF and/or Flt-1.
 15. The composition of claim 2,wherein the ligand is attached to another molecule or compound.
 16. Thecomposition of claim 15, wherein the ligand is attached to an antibody,bispecific antibody, antibody fragment, chimeric antibody, humanizedantibody, human antibody, human antibody fragment, antibody analog, Fcfragment, Fc-binding protein, antibody-binding fusion protein, drug,prodrug, toxin, enzyme, oligonucleotide, radioisotope, immunomodulator,cytokine, hormone, binding molecule, lipid, polymer, micelle, liposome,nanoparticle, or combination thereof.
 17. The composition of claim 16,wherein the ligand is attached to a molecule that binds to a tumorantigen.
 18. The composition of claim 15, wherein the ligand is attachedto a molecule that binds to a disease target.
 19. The composition ofclaim 17, wherein the molecule is a bispecific antibody or fragmentthereof, said antibody or fragment with one binding site for atumor-associated antigen and a second binding site for the PlGF ligand.20. The composition of claim 15, wherein the ligand is covalentlyattached to a monoclonal antibody, said monoclonal antibody with abinding site for a disease target.
 21. The composition of claim 19,wherein the bispecific antibody has a binding site for a tumorassociated antigen selected from the group consisting of A3, antigenspecific for A33 antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15,CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80,HLA-DR, NCA95, NCA90, HCG and its subunits, CEA (CEACAM5), CEACAM6,CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia induciblefactor (HIF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophageinhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA, PSMA,RS5, S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1(IGF-1), Tn antigen, Thomson-Friedenreich antigens, a tumor necrosisantigen, VEGF, ED-B fibronectin, 17-1A-antigen, an angiogenesis marker,an oncogene marker and an oncogene product.
 22. The composition of claim1, wherein the tumor is selected from the group consisting of acutelymphoblastic leukemia, acute myelogenous leukemia, biliary cancer,breast cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colorectal cancer, endometrial cancer, esophagealcancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lungcancer, medullary thyroid, non-Hodgkin's lymphoma, ovarian cancer,pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, andurinary bladder cancer.
 23. The composition of claim 15, wherein thePlGF ligand is a binding peptide and the binding peptide is covalentlyattached to an Fc fragment of an antibody, an Fc-binding protein or anantibody-binding fusion protein.
 24. The composition of claim 23,wherein the PlGF ligand is attached to an Fc fragment of an antibody andthe antibody is IgG1.
 25. A method for inhibiting tumor angiogenesis,metastasis, tumor growth, tumor cell survival and/or cancer cellmotility comprising: a) obtaining a ligand that binds to placenta growthfactor (PlGF); and b) administering the ligand to a subject wherein theligand inhibits tumor angiogenesis, metastasis, tumor growth, tumor cellsurvival and/or cancer cell motility.
 26. The method of claim 25,wherein the ligand is a peptide.
 27. The method of claim 26, furthercomprising identifying the peptide by phage display.
 28. The method ofclaim 26, wherein the peptide comprises at least 12 contiguous aminoacid residues selected from the sequence of BP-1 (SEQ ID NO: 1), BP-2(SEQ ID NO:2), BP-3 (SEQ ID NO:3) or BP-4 (SEQ ID NO:4).
 29. The methodof claim 28, wherein the peptide comprises the sequence of BP-1 (SEQ IDNO:1).
 30. The method of claim 25, wherein the ligand is selected fromthe group consisting of an antibody, antibody fragment, chimericantibody, humanized antibody, human antibody, human antibody fragmentand antibody analog.
 31. The method of claim 25, wherein the subject hasbreast cancer.
 32. The method of claim 26, further comprisingadministering one or more anti-cancer agents in combination with thepeptide.
 33. The method of claim 25, further comprising administering aninhibitor of phosphatidyl inositol 3 kinase (PI3K) or mitogen activatedprotein kinase-kinase 1 (MEK1) to the subject.
 34. The method of claim33, wherein the PI3K inhibitor is Wortmannin or the MEK1 inhibitor isPD98059.
 35. The method of claim 32, wherein the agent is selected fromthe group consisting of a chemotherapeutic agent, radiation therapy,immunotherapy, radioimmunotherapy, localized hyperthermia, laserirradiation and surgical excision.
 36. The method of claim 25, furthercomprising administering one or more anti-angiogenic compounds to thesubject.
 37. The method of claim 36, wherein the compound inhibitsVEGF-mediated angiogenesis.
 38. The method of claim 25, wherein theligand inhibits PlGF-activated cancer cell motility.
 39. A method oftreating a condition relating to angiogenesis comprising: a) obtaining aligand that binds to placenta growth factor (PlGF); and b) administeringthe ligand to a subject wherein the ligand inhibits angiogenesis. 40.The method of claim 39, wherein the condition is selected from the groupconsisting of cancer, hyperplasia, diabetic retinopathy, maculardegeneration, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, rheumatoid arthritis, sarcoidosis, asthma, edema, pulmonaryhypertension, psoriasis, corneal graft rejection, neovascular glaucoma,Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, restenosis, neointima formation after vasculartrauma, telangiectasia, hemophiliac joints, angiofibroma, fibrosisassociated with chronic inflammation, lung fibrosis, deep venousthrombosis and wound granulation.
 41. A method of targeting an agent toa tumor or vascular endothelial cell comprising: a) obtaining a ligandthat binds to placenta growth factor (PlGF); and b) attaching the ligandto the agent; wherein the PlGF ligand binds to tumor and/or vascularendothelial cells.
 42. The method of claim 41, wherein the ligand isselected from the group consisting of BP-1 (SEQ ID NO:1), BP-2 (SEQ IDNO:2), BP-3 (SEQ ID NO:3) and BP-4 (SEQ ID NO:4).
 43. The method ofclaim 41, further comprising administering a bispecific antibody thathas a binding site for the PlGF ligand and a second binding site for atumor antigen.
 44. The method of claim 43, wherein a ligand-antibodycomplex is administered orally or inhalationally and the ligand-antibodycomplex is absorbed through interaction with a neonatal Fc receptortransport system.
 45. A kit comprising a PlGF ligand and a container.46. The kit of claim 45, further comprising a chemotherapeutic agent, abispecific antibody, an anti-angiogenic agent or a combination thereof.47. A fusion protein comprising a PlGF ligand sequence and a secondsequence.
 48. The fusion protein of claim 47, wherein the PlGF ligandsequence comprises at least 12 contiguous amino acids selected from thesequence of BP-1 (SEQ ID NO:1), BP-2 (SEQ ID NO:2), BP-3 (SEQ ID NO:3)or BP-4 (SEQ ID NO:4).